62204

antibody from Invitrogen Antibodies

Targeting: TUBA1C bcm948, MGC10851, MGC14580, TUBA6

Provider product page for 62204

Western blot

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Antibody data

Antibody Data
Antigen structure
References [161]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [25]

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Product number: 62204 - Provider product page
Provider: Invitrogen Antibodies
Product name: alpha Tubulin Monoclonal Antibody (DM1A)
Antibody type: Monoclonal
Antigen: Other
Description: 62204 detects alpha tubulin in human, mouse, rat, bovine, porcine, chicken, amphibian, gerbil and guinea pig samples.
Antibody clone number: DM1A
Concentration: 0.5 mg/mL

TUBA1C protein structure - 62204 shown in red.

Supportive validation

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Experimental details: Western blot analysis was performed on membrane enriched extract (30 µg) of SH-SY5Y (Lane 1), U-87 MG (Lane 2) COLO 205 (Lane 3), HT-29 (Lane 4), PC-3 (Lane 5), MDA-MB-231 (Lane 6), MCF7 (Lane 7), NIH/3T3 (Lane 8), A-431 (Lane9) and Jurkat (Lane10). The blots were probed with Anti-Alpha Tubulin Mouse Monoclonal Antibody (Product # 62204, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~ 55 kDa band corresponding to Alpha Tubulin was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE®12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

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Experimental details: Western blot analysis was performed on whole cell extracts (30µg lysate) of A-431 (Lane 1), COS-7 (Lane 2), MDCK (Lane 3), NIH/3T3 (Lane 4), HT-29 (Lane 5), PC-12 (Lane 6), Neuro-2a (Lane 7), tissue extracts of Mouse Pancreas (Lane 8) and Rat Brain (Lane 9). The blot was probed with Anti- alpha Tubulin Mouse Monoclonal Antibody (Product # 62204, 1µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25µg/mL, 1:4000 dilution). A 55 kDa band corresponding to alpha Tubulin was observed across the cell lines and tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 EphrinB1 has novel MAP characteristics and co-immune precipitates with tubulin Coomasie stained SDS-PAGE gel of MAP spin-down assay showing that microtubule associated protein, MAP2, pellets with microtubules but a non-MAP, bovine serum albumin (BSA), does not. S: soluble fraction; P: pellet, 1: microtubules only; 2: MAP2+ microtubules; 3: BSA+ microtubules (A). Western blot analysis of microtubule spin-down of EphrinB1DeltaED; S: soluble fraction; P: pellet (B). Western blot analysis of mitotic, soluble and insoluble fractions that have been immunoprecipitated for EphrinB1 (C).

Supportive validation

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Experimental details: Fig 1 Microtubule disruption dephosphorylates p120 and stimulates cadherin-based adhesion in Colo 205 cells. (A-F) Brightfield microscopy of non-adhesive and adhesion-activated Colo 205 cells. Scale bar = 50 mum. (A-C) Parental or p120 knockdown cells were treated with either 10 muM nocodazole or an equal volume of DMSO for 1.5 hours. (D-F) Parental cells were pre-treated with 10 muM taxol for 2 hours then treated with 2 mug/mL 19A11 activating E-cadherin mAb for 4 hours. (G) Colo 205 cells expressing EB1-EGFP were treated with various concentrations of nocodazole and imaged at 10 minutes via fluorescent confocal microscopy. Scale bar = 5 mum. The same cells were then imaged by brightfield microscopy (scale bar = 50 muM) after 2 hours of treatment to examine the extent of adhesion activation, then fixed in methanol and immunostained for alpha-tubulin to examine the extent of microtubule disruption (scale bar = 5 mum). (H) Cells were treated for 1.5 hours with various concentrations of nocodazole then lysed in 1% triton x-100. Samples were resolved on 6% SDS-PAGE with 20 muM Phos-tag reagent then visualized via Western blotting. Red stars = higher molecular weight in non-activated samples, cyan stars = lower molecular weight in activated samples.

Supportive validation

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Experimental details: Figure 4 Analysis of muscle quality in vitamin D sufficient and insufficient mice . To assess impacts of vitamin D insufficiency on muscle biology, tissues were harvested following 12 months of sufficient (STD) or insufficient (LOW) supplementation. Quadriceps muscle was then analyzed with NADH staining ( A ) allowing for quantification of the cross-sectional area (CSA) of light stain fibers that corresponds to fast twitch fibers, n= 5, 4 respectively ( B ). Gastrocnemius muscle was analyzed by differential centrifugation to determine myofibrillar, sarcoplasmic, and mitochondrial protein content, n= 6 ( C ). Atrogin-1 expression was also determined in gastrocnemius muscle with western blotting and relative expression (atrogin / tubulin) was quantified using ImageJ software, n= 6 ( D ). Mitochondrial biomass ( E ) and activity ( F ) in soleus muscle, n= 6, were determined by quantitative PCR and biochemical assays, respectively. Statistical significance indicated by ""*"" p < 0.05 and ""ns"" indicating non-significance.

Supportive validation

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Experimental details: Fig. S2 YK-4-279 induces mitotic arrest and apoptosis. (A) Quantification of percentage of cells at various phases of cell cycle after treatment with DMSO or YK-4-279 for 24 h. Error bars represent +-SD of three independent replicates. ** P < 0.01, *** P < 0.001. Unpaired Student t-test was used to assess the significance of difference between the control and treated samples. (B) SK-N-BE(2)-C and CHP-212 were treated with DMSO (D) or two doses of YK-4-279 for 24 h and cell lysates were harvested in RIPA buffer. Blots were probed for several cell cycle and cell death markers. All neuroblastoma cell lines showed increase of Histone H3 (Ser10), cleaved PARP and active caspase-3. Actin was used as a loading control. (C) Immunoblots demonstrating that YK-4-279-induced cell death of SK-N-AS and GIMEN cells is rescued by QVD treatment. Note decreased cleaved caspase-3 and cleaved PARP after QVD treatment. (D) Immunoblotting for acetyl-alpha-tubulin (Lys40) (indicator of tubulin microtubule stabilization) and phospho-histone H3(ser10) (pHH3). alpha-tubulin and actin were used as loading controls. (E) Knockdown of wildtype p53 in GIMEN and assessment of YK-4-279 activity. Cells were reverse transfected with either control siRNA or p53 siRNA. After 72 h of incubation whole cell lysates were harvested for western analysis and probed for mitotic and apoptotic markers. Fig. S2

Supportive validation

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Experimental details: Fig. S3 Localization of key mitotic kinases in early mitosis. GIMEN cells were treated with YK-4-279 and stained with Aurora A, PLK1 and alpha-tubulin antibodies. Chromatin was visualized using DAPI. (A) Aurora A localised to centrosomes and spindle tubules in both DMSO and YK-4-279 indicating no abnormalities in sub-nuclear distribution. (B) PLK1 staining showing a similar pattern. Tubulin staining revealed appearance of normal astral microtubules. Fig. S3

Supportive validation

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Experimental details: Figure 3. Genome editing of rosetteless enables targeted disruption of multicellular development in S. rosetta. ( A ) An engineered mutation in rosetteless introduces a premature termination sequence (PTS) to knockout the expression of rosetteless . The rosetteless gene (exons shown as numbered black boxes, connected by introns) encodes a secreted protein (SS denotes the signal sequence for secretion) with two C-type lectin domains (CTL1 and CTL2) and two carboxy-terminal repeats (RP1 and RP2). A forward genetic screen () identified a mutation, rtls tl1 , in which a T to C transition in the seventh intron disrupts splicing and knocks out rosetteless expression. To increase the likelihood of disrupting rosetteless function with genome editing, we designed the rtls PTS1 mutation that introduces a PTS (green), with a poly-adenylation sequence and stop codons in each reading frame, into the fourth exon of the gene. ( B ) The genotypes of strains established from genome-editing confirm that rosetteless and rpl36a incorporated the designed mutations. To enrich for genome-edited cells, Sp Cas9 RNPs and repair templates for introducing rpl36a P56Q ( Figure 2--figure supplement 2 ) and rtls PTS1 were simultaneously delivered into S. rosetta . Afterward, cycloheximide resistant cells were clonally isolated and screened for cells that did not develop into rosettes in the presence of RIFs. The genotypes of rtls PTS1 rpl36a P56Q , and rpl36 P56Q confirmed that strains established from gen

Supportive validation

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Experimental details: Figure 1 ( A , B ) Confirmation of efficient siRNA-mediated knockdown of NRAS expression (siNRAS) at mRNA ( A ) and protein ( B ) level with RT-qPCR and Western blot, respectively, at 72 h post transfection. Two siRNAs were used (siNRAS#1 and siNRAS#2; for sequences, see Materials and Methods). The expression of HRAS mRNA ( A ) was unaffected by siNRAS treatment, demonstrating its specificity. In ( A ), mRNA expression in control-transfected (dark-grey bars) and siNRAS-treated (white and light-grey bars) hPheo1 cells is presented as fold change (2 -DeltaDeltaCT ), with results from three independent experiments given as mean +- standard deviation, and p values calculated with student's T -test indicated as * (0.001

Supportive validation

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Experimental details: Figure 4 Naive ES Cells Remain Connected after Division (A) Representative confocal image of a naive ES cell colony stained for alpha-tubulin (black, inverted contrast). A maximum Z projection is shown. Scale bar: 10 mum. (B) Representative time-lapse of a fluorescence recovery after photobleaching (FRAP) experiment in ES cells expressing cytoplasmic GFP, the two cells at the top are sister cells. GFP intensity levels are displayed. Photobleaching is performed at 0 s in the sister cell on the right (red lightning bolt). GFP intensity is then monitored in the bleached cell (dark gray box), the sister cell (red box) and an unconnected neighbor (blue box). One Z plane is shown, time in seconds, scale bar: 10 mum. (C) Left panel: plot showing the mean GFP intensity over time (smoothed over a 4 point, ~4.5 s, window) in the boxes in the cells depicted in (B) Dotted lines highlight minimum fluorescence levels for the two unbleached cells. Right panel: transmitted light image and fluorescent Z projection (inverted contrast) of the 3 cells displayed in (B) and labeled with SIR-tubulin prior to the FRAP experiment, highlighting the tubulin bridge connecting the two cells at the top. (D) Dot plot showing the minimum GFP levels in the sister cell of the ES cell where GFP was bleached (red) and for an unconnected neighboring cell at a similar distance (blue). Mean and standard error of the mean are plotted. N = 3. (E) Dot plot showing the minimum GFP levels following photobleaching in on

Supportive validation

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Experimental details: Figure 5 Abscission Duration Decreases during Naive Pluripotency Exit (A) Representative confocal images of cells at different stages of naive pluripotency exit and stained for alpha-tubulin (white) and CRIK (magenta). Pink arrowhead, example of a bridge with a CRIK spot; white arrowhead, example an isolated CRIK spot, suggesting a midbody remnant. Cells are cultured on laminin to facilitate the visualization of the bridges. Scale bars: 10 mum. (B) Dot plot showing the fraction of cells with bridges (number of bridges divided by number of cells in a given analysis frame) in H2B-RFP ES cells and during naive pluripotency exit on laminin. Mean and standard error of the mean are shown. N = 2. (C) Representative time-lapses of a colony of ES cells expressing H2B-RFP and labeled with SIR-tubulin (black, inverted contrast, maximum Z projection across the colony is shown). Time in min. 0 min: end of cytokinesis. Top, naive cells; Middle and bottom, cells 25 and 45 h after induction of naive pluripotency exit, respectively. Red arrows: abscission. Scale bars: 10 mum. (D) Dot plot showing the duration of abscission for HeLa cells expressing tubulin-GFP dividing on elongated line micropatterns to standardize cell shape (blue) and for naive ES cells and cells exiting naive pluripotency labeled with SIR-tubulin (gray and black dots). Mean and standard error of the mean are shown. N = 3.

Supportive validation

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Experimental details: Figure 6 Bridge Thinning and CHMP4B Recruitment Accompany Shortening of Abscission Duration during Naive Pluripotency Exit (A) Representative confocal images showing cells stained for alpha-tubulin (white) and CHMP4B (red) during naive pluripotency exit. Inset: zoom of representative bridges. Scale bars: 10 mum. (B) Dot plot showing the fraction of bridges displaying a CHMP4B spot near the bridge center in cell colonies during naive pluripotency exit on laminin. Mean and standard error of the mean are shown. N = 2. (C) Representative electron microscopy images of bridges connecting two naive cells (left) and two cells after 48 h in differentiation medium (right). Scale bars: 1 mum. (D) Dot plot showing bridge width measured from electron microscopy images of ES cells and cells exiting naive pluripotency. Mean and standard error of the mean are plotted. N = 2. (E) Dot plot showing the size of the electron dense midzone of the bridge, corresponding to the midbody, measured from electron microscopy images of ES cells and cells exiting naive pluripotency. Mean and standard error of the mean are plotted. N = 2. (F) Representative time-lapses of tubulin bridges in ES cells (top) or cells after 24 h in differentiation medium (bottom) labeled with SIR-tubulin (black, inverted contrast), Z projections are shown. Time in min. The pink arrowheads point to the bridges. Scale bars: 10 mum. (G) Time course of bridge width after cytokinesis for naive cells (black) and cells after 24 h in di

Supportive validation

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Experimental details: Figure 7 Abscission Regulates Exit from Naive Pluripotency (A) Confocal images of H2B-RFP ES cells treated with scrambled siRNA (SCR, top) or siRNA against Alix (bottom) for 24 h in 2i/LIF, then plated on laminin in 2i/LIF or N2B27 (24 and 48 h exit), and stained for alpha-tubulin (white) and CRIK (magenta). A maximum Z projection over the volume of the colony is shown. Scale bars: 10 mum. (B) Dot plot showing the fraction of cells with bridges (number of bridges divided by number of cells in a given analysis frame) in ES colonies pre-treated with siRNA Scrambled ( SCR , gray) or Alix (orange) for 24 h in 2i/LIF, during naive pluripotency exit on laminin. Mean and standard error of the mean are shown. N = 2. (C) Dot plot representing the number of colonies surviving in a clonogenicity assay (see Figure 2 A) for ES cells treated with siRNA Scrambled ( SCR , gray) or Alix (orange) for 24 h in 2i/LIF, then placed in differentiation media for 24 h. The mean and standard deviation are shown. N = 6. (D) Schematic representation of the laser ablation experiment: 5 h after inducing naive pluripotency exit, a tubulin bridge connecting two cells is ablated using a pulsed laser (red thunderbolt), and REX1-GFP levels in the 2 cells (dark gray and yellow) are monitored. (E) Representative example of a bridge laser ablation experiment in cells labeled with SIR-tubulin (inverted contrast). Ablation is highlighted with a red thunderbolt and the location of the ablated bridge and destroyed mi

Supportive validation

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Experimental details: Figure 5 Representative western blot images of total protein extracted from two arteriovenous malformation-derived primary cell lines probed for OCT4 (A) , SOX2 (B) , NANOG (C) , KLF4 (D) , c-MYC (E) , and alpha-tubulin (F) , with appropriate molecular weights labeled.

Supportive validation

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Experimental details: Figure 4 Differentiation of mammary gland epithelial cells in control ( Ctr ) and Miz1 Delta POZ tissue and in HC11 cells with reduced levels of Miz1. ( A ) Gene expression of the milk proteins alpha-casein, ss-casein and whey acidic protein (Wap), measured by quantitative RT-PCR, in control and Miz1 Delta POZ mammary gland tissue (DeltaPOZ). ( B ) Immunoblot analysis for ss-casein from mice with the indicated phenotype. Each lane represents an individual animal. ( C ) Immunostaining with an antibody against milk proteins in the acini from control and Miz1 Delta POZ mammary glands. ( D ) Sudan III staining was performed on cryosections from lactating mammary glands of Ctr and MizDeltaPOZ mice (n = 4 per genotype). Data from A to D was obtained from lactation day 6 samples. ( E ) Time course of growth and differentiation as performed in the experiments with HC11 cells. EGF: epidermal growth factor; DIP: differentiation media containing dexamethasone, insulin and prolactin. Time points indicated correlate to the time points in ( G ) and ( H ). ( F ) HC11 cells were stably transfected with scrambled short hairpin (sh) RNA (shscr), a Miz1 shRNA and a vector expressing Miz1 as a positive control. The film was exposed 1 and 5 minutes, respectively. ( G ) PCR and ( H ) Western blots revealed a down-regulation of ss-casein when Miz1 concentration is decreased. Numbers indicated in ( B ) and ( H ) are fold changes of band intensities obtained by densitometry (see Materials and Methods

Supportive validation

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Experimental details: Figure 5 Stat5 phosphorylation is reduced in Miz1 Delta POZ mammary glands and in shMiz1 HC11 cells. ( A ) Analysis of Stat5a/b mRNA expression in Miz1 Delta POZ (DeltaPOZ) and control mammary gland tissue ( Ctr ). ( B ) Immunoblot analysis of Stat5 in wildtype and Miz1 Delta POZ mammary glands. ( C ) Immunohistochemical staining of pStat5 in the mammary gland tissue from wildtype and Miz1 Delta POZ animals. ( D ) Western blots from HC11 cells stably transfected with a scrambled short hairpin (sh) RNA (shscr) or a Miz1 shRNA (see also Fig. 4 E and F ). Numbers indicated are fold changes of band intensities obtained by densitometry (see Materials and Methods). Quantitative RT-PCR for the prolactin receptor ( E ; Prlr ), the Supressors of cytokine signalling ( Socs ) 1, 2 and 3 and caveolin-1 ( F ; Cav1 ), and for ErbB4 ( G ). Lactation day 6 samples were used in all in vivo experiments. Scale bar in C: 50 um.

Supportive validation

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Experimental details: Figure 2--figure supplement 1. Representative phospho-LAT Y191 western blots. ( A ) Wild type or Itk-deficient 5C.C7 T cells were activated with CH27 B cells and 10 um MCC in the presence or absence of 10 ug/ml anti-CD80 plus anti-CD86. At the given time points (in minutes) T cell:APC cell extracts were blotted for LAT phosphorylation at Y191. Blots were stripped and reblotted with an anti-alpha tubulin antibody as a loading control. Phospho-LAT Y191 band intensities as normalized to loading control are given under the phospho-LAT blots. Molecular weights of marker bands (in kD) are given at the right. One representative blot of eight is shown. ( B ) 5C.C7 T cells were activated with CH27 B cells and 10 um MCC for the indicated times (in minutes) with the additional controls of stimulation with pervanadate ('+') and 5C.C7 T cell CH27 B cell interactions in the absence of MCC peptide ('-'). One representative blot of three is shown.

Supportive validation

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Experimental details: Figure 4--figure supplement 1. Patterning of isolated targeting domains and interface recruitment of LAT-GFP and spatially targeted version thereof. ( A ) The graphs give the percentage of cell couples that displayed accumulation of the isolated targeting domains, LAT V3, LAT Vav, and LAT PLCdeltaPH as indicated, with the indicated patterns as in Figure 2B relative to tight cell couple formation for wild type 5C.C7 T cells activated with CH27 B cell APCs (10 uM MCC peptide). 30-57 cell couples from 2 to 3 independent experiments were analyzed per condition, 133 total. ( B ) Wild type 5C.C7 T cells were transduced to express LAT-GFP and sorted for LAT-GFP positive (LAT-GFP pos) and LAT-GFP negative (LAT-GFP neg) T cells or left non-transduced. Cell extracts were blotted for LAT (green) and alpha tubulin (red) as a loading control. One representative blot of three is shown. ( C ) LAT band intensities were quantified and are shown relative to endogenous LAT in the non-transduced cells. The GFP intensity used to sort LAT-GFP-positive 5C.C7 T cells corresponds to 6*10 5 molecules per cell ( Singleton et al., 2009 ). ( D, E ) For the quantification of the accumulation of LAT and targeted versions thereof at the T cell:APC interface we applied a computational image quantification as recently described in detail ( Roybal et al., 2016 ). We identified a core region ( B ) of sensor enrichment as defined as the 10% most fluorescent voxels of the average probability distribution across a

Supportive validation

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Experimental details: Figure 7. TTC17 is required for the polarized arrangement of Golgi cisternae and posttranslational modifications. (A-C) Control HeLa cells (A) and cells depleted of CCDC157 (B) or TTC17 (C) were incubated in the presence or absence (DMSO) of 500 ng/ml nocodazole for 2 h, and analyzed by immunofluorescence microscopy with anti-TGN46 and anti-GM130 antibodies. Depolymerization of the microtubule cytoskeleton was monitored with an anti-alpha-tubulin antibody. Nuclei were stained with DAPI. Scale bars, 10 um. Insets from A, B, and C, show Golgi stack dispersion after nocodazole treatment. Intensity profile graphs of TGN46 and GM130 colocalization derived along the lines indicated in the confocal micrograph inserts. (D) Pearson's correlation coefficient of colocalization between TGN46 and GM130 from confocal micrographs acquired for control cells, and cells depleted of CCDC157 or TTC17 after nocodazole treatment ( n = 24; two-tailed Student's t test for comparison to control condition siCtr; ***, P < 0.001; ns, statistically nonsignificant). (E) Total cell lysates of control HeLa cells and cells depleted of TTC17 or CCDC157 were analyzed by immunoblotting with anti-LAMP2 and anti-alpha-tubulin antibodies. Results are representative of three independent experiments.

Supportive validation

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Experimental details: Figure 5 Western blots demonstrating the expression of PRR at 21 kDA ( A , blue) and ATIIR1 at 42 kDa ( C , blue), but not ACE ( B , blue), or ATIIR2 ( D , blue). Bands for alpha-tubulin ( A-D , green) confirmed efficient transfer and approximate equivalent protein loading for all WHO grade I meningioma samples examined.

Supportive validation

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Experimental details: Figure 7. Postnatal knockdown of Dcx slightly increases dendritic spine head and neck widths. ( A ) High magnification confocal images of dendritic spines from mismatch and Dcx-pKD neurons co-electroporated with mCherry. Scale bar = 2 mum. ( B ) Quantification of the density of spines for 100 mum of dendrites in mismatch and Dcx-pKD neurons. Values are given as mean +- SEM. Each dot represents a neuron. ( C - E ) Bar graphs and scatter dot plots illustrating the mean dendritic spine length ( C ), head width ( D ) and neck width ( E ) in mismatch and Dcx-pKD neurons. Values are given as mean +- SEM. Each dot represents a spine. Dotted line frames hold higher magnifications of the initial bar graphs. ( D , E ) Mann-Whitney test, P -value < 0.001. ( F ) Western blot of the synaptosomal and cytosolic fractions isolated from cortices of P10, P15, and P20 non-electroporated rats. (Top panels) Detection of PSD-95 (top) and Dcx (bottom). (Bottom panels) Detection of alpha-tubulin for control.

Supportive validation

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Experimental details: Figure 1 Overexpression of SETDB1 in Melanoma Cell Lines Resulted in the Activation of Protumorigenic-Secreted Factors (A) Histogram plot showing microarray expression data (shown as log 2 fold change) of genes encoding for protumorigenic-secreted factors (THBS1, SCG2, MMP1, MMP3, IL-6, IL-8, CCL2, and WNT5A) in HT 144-SETDB1 OE cells compared to EV cells. (B) Quantitative real-time PCR analysis of the CRS factor-encoding genes SCG2 , MMP3 , IL8 , CCL2 , and WNT5A expression 5. (C) Secretome analysis of HT 144 EV and -SETDB1 OE cell supernatant. Right panel: blots showing the signals of 55 different secreted proteins detected in the supernatant of cultured cells; signals of MMP8 (1), CXCL16 (2), CCL2 (3), and THBS1 (4) are highlighted. Left panel: quantification of mean pixel density related to MMP8, CXCL16, CCL2, and THBS1 signals. Number or replicates: 3. (D) SCG2 immunofluorescence detection in C32 EV (control) and -SCG2 OE cell lines. SCG2-overexpressing cells showed a strong SCG2 signal accumulation at granule structures (indicated by arrows). DAPI stained the nuclei. Scale bars, 20 mum. (E) Tissue microarray analysis (TMA) of a cohort of melanoma patients showing that SETDB1 expression positively correlated with SCG2 expression in melanoma metastases tissue samples. SETDB1 and SCG2 correlation plot is shown. Number of analyzed patients' samples (n), Spearman correlation coefficient (r), and p value are reported. (F) Kaplan-Meier survival analysis of patients with melano