33-2000

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on nuclear enriched cell extracts of HeLa (Lane 1), HEK-293 (Lane 2) and K-562 (Lane 3). The blots were probed with Anti- Lamin B1 Mouse Monoclonal Antibody (Product # 33-2000, 2µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjµgate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~ 66 kDa band corresponding to Lamin B1 was observed across all cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), 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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Lamin B1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR711025_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Lamin B1 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa Lamin B1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Lamin B1 Monoclonal Antibody (L-5) (Product # 33-2000, 1:250 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Lamin B1. An uncharacterized band was observed in HeLa Cas9 sample at ~45 kDa.

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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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

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

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

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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 3: ZO-2 triple mutant S257E/S259E/S261E and the single mutants S257E and S257A arrive at the nucleus with a delay. (A) Percentage of cells with nuclear HA-ZO-2 as a function of time. The percentage of cells with nuclear ZO-2 was determined by immunofluorescence using an anti-HA antibody. Monolayers were fixed at the indicated times. Time 0 corresponds to 6 h after transfection. Experiments were done with cells transfected with full-length HA-ZO-2 (WT), the triple mutant S257E/S259E/S261E and single mutants S257E and S257A. Results from three independent experiments. In each experiment 100 transfected cells were evaluated per experimental point. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 with respect to ZO-2 WT as assessed by two-way ANOVA followed by Bonferroni's post hoc test. (B) Detection by Western blot of HA-ZO-2 present in plasma membrane and nuclear fractions derived from cells 24 h after being transfected with HA-ZO-2 WT or the mutants S257A and S257E. Lamin B1 and Na + -K + -ATPase beta1 subunit antibodies were used as positive controls of nuclear and plasma membrane fractions, respectively.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Proof-of-concept for therapeutic efficacy and mechanism of s50-TBG-RNAi-CK2 in orthotopic and metastatic prostate cancer xenograft tumors. ( a ) Primary tumor mass and lymph node tumor volumes are shown following s50-TBG-RNAi-CK2 treatments at 33 and 330 ng/kg compared to vehicle. Means +- SE are presented (TBG-RNAi-CK2 n = 4; Vehicle n = 5). * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 INPP5F inhibits STAT3 phosphorylation and nuclear translocation. (A) Immunoblot analysis shows that INPP5F overexpression in glioblastoma cells (GSC923, GSC1228, and U87) inhibits phosphorylation of STAT3 (Y705) and INPP5F knockdown (GSC211 and GSC827) enhances its phosphorylation. (B) Immunoblot analysis of nuclear [N] and cytosol [C] fractions reveals that overexpression of INPP5F inhibits STAT3 nuclear translocation while INPP5F knockdown enhances the nuclear translocation of STAT3. (C) Control and INPP5F overexpressing GSC923 cells were treated with IL-6 (50 ng/ml) for 5 and 10 min, and immunoblot was performed with anti-p-STAT3 (Y705). Immunoblot analysis shows that INPP5F inhibits IL-6 induced phosphorylation of STAT3. (D) Confocal microscopy analysis verified that overexpression of INPP5F decreases STAT3 phosphorylation and nuclear translocation compared to control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Pref-1 induces nucleocytoplasmic translocation of FOXO1 and PDX1 in PANC1 cells. ( a ) Immunoblot analyses and their quantification of ERK1/2, phosphorylated FOXO1, FOXO1, and PDX1 at day 2 after transfection with either pSPORT6-hDLK1 as Pref-1 or pCMV as control. Cells were treated with 25 muM PD98059 or 20 muM LY294002. n = 3, * P < 0.05 vs pCMV, # P < 0.05 between groups (means +- SE). ( b ) Protein levels of FOXO1 and PDX1 in the cytoplasmic and nuclear fractions at the indicated times after Pref-1 transfection, and their quantification. n = 3, ** P < 0.01 vs day 0 (means +- SE). ( c ) Immunostaining of PDX1 and FOXO1 at day 6 after transfection with either pSPORT6-hDLK1 or pCMV. Scale bar, 5 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 8: PUGNAc-induced stabilization of O -GlcNAcylation promotes nuclear exportation and degradation of ZO-2. Sparse cultures of MDCK cells were treated or not for 16 h with 100 muM PUGNAc or 100 muM PUGNAc plus 30 mM MG132. (A) Western blot detection of O -GlcNAc in MDCK cell extracts treated or not with PUGNAc (left). PUGNAc induces the disappearance of ZO-2 from the nuclei and cellular borders. Monolayers were fixed and processed for immunofluorescence with an antibody against ZO-2. The quantitative analysis of ZO-2 fluorescence signal intensity in the nucleus and cytoplasm was made with ImageJ particle analysis (bottom right). Results from three independent experiments. * p < 0.05 and ** p < 0.01 as assessed by two-way ANOVA followed by Bonferroni's post hoc test. (B) Treatment with PUGNAc decreases nuclear ZO-2. Left, Western blot against ZO-2 of nuclear (N), cytoplasmic (C), and plasma membrane (PM) fractions derived from cells treated or not with PUGNAc or PUGNAc plus MG132. Antibodies against beta 1 -Na + -K + -ATPase, actin, and lamin B1 were used as positive controls of PM, C, and N fractions, respectively. Right, densitometric analysis of the Western blot. Results from three independent experiments. * p < 0.05, ** p < 0.01, and **** p < 0.0001 as assessed by two-way ANOVA followed by Bonferroni's post hoc test. (C) Treatment with PUGNAc ablates the expression of O -GlcNAcylated ZO-2. The Duolink in situ immunoassay was done with an antibody against ZO-

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Effects of melittin on PgLPS-induced activation of NF-kappaB signaling pathway, Akt, and ERK1/2. Representative Western blot images show the effects of PgLPS and melittin on the activation of cytosolic NF-kappaB inhibitor (IkappaB), nuclear NF-kappaB ( A ); Akt, and ERK1/2 ( B ). The bar graphs show quantitative signal intensities of the proteins after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Lamin B 1 , Akt, and ERK 1/2, respectively. -: untreated, +: treated. * p < 0.05 compared to the untreated group. + p < 0.05 compared to the PgLPS group. ( C ) Representative immunofluorescence images show the effects of PgLPS and melittin on the activation of NF-kappaB (labeled with Alexa Fluor 647, red) in HaCaT cells. The nuclei were labeled with Hoechst 33342 (blue). beta-actin was labeled with Alexa Fluor 488 (green). PgLPS: 0.1 mug/mL of P. gingivalis lipopolysaccharides, Melittin: 1 mug/mL of melittin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4: ZO-2 is sequestered in the cytoplasm by 14-3-3 in the LC condition. (A) cZO-2 and hZO-2 sequences, respectively, have 25 and 22 putative 14-3-3 binding sites, 11 (cZO-2) and 10 (hZO-2) of which are located at the U2 segment. Scheme showing the molecular organization of ZO-2. PDZ, PDZ domain; SH3, Src homologous 3 domain; GK, guanylate kinase domain; Acidic, acidic region; PR, proline-rich segment; ABR, actin binding region; U, unique region; TEL, PDZ binding motif; NLS, nuclear localization signal; NES, nuclear exportation signal; vertical lines, putative 14-3-3 binding sites; blue letters and numbers, putative 14-3-3 binding sites located within an SR motif. (B) The inhibition of 14-3-3 with BV02 induces the appearance of ZO-1 and ZO-2 at the cell borders of cells cultured in LC. MDCK cells were plated at confluent density in NC media and after 1 h were washed 5x with PBS without Ca 2+ and transferred to LC media with 50 muM BV02 or vehicle only (DMSO 0.25%, control). After 20 h the monolayers were fixed and processed for immunofluorescence with antibodies against ZO-1, ZO-2, claudin-1, occludin, and cingulin. Top panel, representative images from three independent experiments. Nuclei were stained in blue with DAPI. Bar, 25 mum. Bottom panel, quantification of ZO-1 and ZO-2 fluorescence staining at the cell borders. Statistical analysis done with Student's t test, **** p < 0.0001. Results obtained from six optical fields in each experimental condition. (C) The cell

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 12: ZO-2 colocalizes with 14-3-3 in the nucleus, and residue S261 of cZO-2 is crucial for the nuclear importation the protein. (A) Left panel, percentage of cells with nuclear HA-ZO-2 as a function of time after transfection, determined by immunofluorescence using an anti HA antibody. Monolayers cultured in NC media were fixed at the indicated times. Time 0 corresponds to 6 h after transfection. Experiments were done with cells transfected with HA-cZO-2 WT, HA-cZO-2 S261A, and HA-cZO-2 T248A. Results are from three independent experiments. In each experiment, 100 transfected cells were evaluated per experimental point. Statistical analysis done with two way ANOVA followed by Bonferroni's multiple comparison test. cZO-2 S261A and HA-cZO-2 T248A were compared against HA-cZO-2 WT; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (B) Western blot against HA-ZO-2 present in nuclear, cytoplasmic, and plasma membrane fractions of cells at times 0 and 24 h of transfection with HA-cZO-2 WT and HA-cZO-2 S261A. Antibodies against lamin B1, Na + ,K + -ATPase beta1 subunit, and GAPDH were employed as markers of nuclear, plasma membrane, and cytoplasmic fractions, respectively. Right panel, overexposed Western blot. (C) The reduced cellular content of HA-cZO-2 mutants T248A and S261A in comparison to WT HA-cZO-2 in LC cultured cells, recovers after proteasome inhibition with MG132. Western blot of cellular lysates derived from monolayers transfected with WT HA-cZO-2 or the m

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 2 The EGFR localization in nuclear and cytoplasmic fractions after treatment of A431 cells with sHB-EGF. Non-nuclear and nuclear cellular extracts were subjected to western-blot analysis after sHB-EGF treatment in the absence or presence of EGFR tyrosine kinase inhibitor AG1478. Down-regulation of EGFR activity by kinase inhibitor AG1478 suppressed the EGFR nuclear localization in A431 cells under sHB-EGF treatment.