- 700712 - Invitrogen Antibodies MAP1LC3B antibody

Submitted references Degradation of lipid droplets by chimeric autophagy-tethering compounds.

Fu Y, Chen N, Wang Z, Luo S, Ding Y, Lu B
Cell research 2021 Sep;31(9):965-979

Acid ceramidase controls apoptosis and increases autophagy in human melanoma cells treated with doxorubicin.

Lai M, Amato R, La Rocca V, Bilgin M, Freer G, Spezia P, Quaranta P, Piomelli D, Pistello M
Scientific reports 2021 May 27;11(1):11221

Role of prostaglandin E2 receptor 4 in the modulation of apoptosis and mitophagy during ischemia/reperfusion injury in the kidney.

Ding C, Han F, Xiang H, Wang Y, Dou M, Xia X, Li Y, Zheng J, Ding X, Xue W, Tian P
Molecular medicine reports 2019 Oct;20(4):3337-3346

Codonopis bulleynana Forest ex Diels inhibits autophagy and induces apoptosis of colon cancer cells by activating the NF-κB signaling pathway.

Luan Y, Li Y, Zhu L, Zheng S, Mao D, Chen Z, Cao Y
International journal of molecular medicine 2018 Mar;41(3):1305-1314

Mannich Curcuminoids as Potent Anticancer Agents.

Gyuris M, Hackler L Jr, Nagy LI, Alföldi R, Rédei E, Marton A, Vellai T, Faragó N, Ózsvári B, Hetényi A, Tóth GK, Sipos P, Kanizsai I, Puskás LG
Archiv der Pharmazie 2017 Jul;350(7)

A quantitative TR-FRET plate reader immunoassay for measuring autophagy.

Hancock MK, Hermanson SB, Dolman NJ
Autophagy 2012 Aug;8(8):1227-44

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of LC3B was performed by loading 30 µg of Mouse Brain (lane1) and Rat Brain (lane2) tissue lysate using NuPAGE® Novex® 4-12% Bis-Tris gel (Product # NP0322BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. LC3B was detected at ~15 kDa using LC3B Recombinant Rabbit Monoclonal Antibody (Product # 700712) at 2-3 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and 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: Western blot was performed using Anti-LC3B Recombinant Rabbit Monoclonal Antibody (2H30L32)(Product # 700712) and a 14kDa band corresponding to LC3B was observed across cell lines and tissue extracts tested and increased upon Chloroquine diphosphate treatment. Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with Chloroquine diphosphate (50uM for 12 hr) (Lane 2), HCT 116 (Lane 3), HCT 116 treated with Chloroquine diphosphate (50uM for 12 hr) (Lane 4), NIH/3T3 (Lane 5) and tissue extracts of Mouse Brain (Lane 6) and Rat Brain (Lane 7) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2 ug/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MAP1LC3B in rat brain cell lysate (lane 1) and mouse brain cell lysate (lane 2) (30 µg/each lane) using a MAP1LC3B recombinant rabbit monoclonal antibody (Product # 700712) at a dilution of 2.5 µg/mL. NBT/BCIP was used as the substrate (Product # WB7105).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MAP1LC3B in HeLa cell lysate treated with Chloroquine (50 uM, 16 hrs) (30 µg/lane) using a MAP1LC3B recombinant rabbit monoclonal antibody (Product # 700712) at a dilution of 2.5 µg/mL. NBT/BCIP was used as the substrate (Product # WB7105).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of LC3B was done on 70% confluent log phase SHSY5Y cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with LC3B Recombinant Rabbit Monoclonal Antibody (Product # 700712) at 2 µg/mL and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of MAP1LC3B showing staining in the cytoplasm of paraffin-embedded human astroglioma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with MAP1LC3B (2H30L32) Monoclonal antibody (Product # 700712) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MAP1LC3B showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with MAP1LC3B (2H30L32) Monoclonal antibody (Product # 700712) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MAP1LC3B showing staining in the cytoplasm of paraffin-embedded rat brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with MAP1LC3B (2H30L32) Monoclonal antibody (Product # 700712) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of LC3B was done on SH-SY5Y cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with ABfinity™ LC3B Recombinant Rabbit Monoclonal Antibody (700712, red histogram) or with rabbit isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin immunoprecipitation analysis of MAP1LC3B was performed using cross-linked chromatin from 1 x 10^6 HCT116 human colon carcinoma cells treated with serum for 0, 15, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a MAP1LC3B rabbit monoclonal antibody (Product # 700712). Chromatin aliquots from ~1 x 10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify the promoter region of human UBE2B, or the imprinting control region (ICR) of the human H19 locus. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the human UBE2B and H19 loci are shown above the data where boxes represent exons (grey boxes = translated regions, white boxes = untranslated regions), the zigzag lines represent introns, and the straight line represents upstream sequence. Regions amplified by UBE2B and H19 primers are represented by black bars. Data courtesy of the Innovators Program.

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: Fig. 4 Formation of the LC3B-ATTEC-LD ternary complex and colocalization between LC3B and LD. a Measurements of the C1-LC3B, C2-LC3B, C3-LC3B, C4-LC3B, SIII-LC3B and SIV-LC3B binding affinity by MST. Submicromolar to micromolar K d values were observed for the interaction between LC3B and LD*ATTECs (C1-C4), but not LD probes (SIII and SIV). b Left: schematic illustration of the measurements of the ternary complex formation using modified ELISA assays; Right: the blank-corrected ELISA signals of the indicated samples ( n = 3, independent assay wells). All samples were added with recombinant purified LC3-GST for the final detection with the GST antibody, and the wells containing recombinant purified GST alone were used as the blank control. c Representative images and quantifications of mCherry-LC3B-transfected MEFs showing LDs (stained with BODIPY 493/503, green), autophagosomes (LC3B puncta, red) and nuclei (DAPI, blue). LD*ATTECs (C1-C4) but not the control compounds (all at 5 muM) induced significant partial colocalizations of LD and autophagosomes. The percentage of LDs that are partially colocalized with autophagosomes in each sample was analyzed by visual counting in a blinded manner. The replicate number indicates the number of fields from 2 independent batches of transfections. d Similar to c , but in LAMP1-mCherry-transfected MEFs showing colocalization between LDs and lysosomes (LAMP1). Bars indicate means +- SEM. ns, P > 0.05; $ P < 0.0001 by one-way ANOVA (the F an

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Immunofluorescence images of LC3B-II in HCT116 and SW480 cells. Cells were treated with cb FeD-containing serum solutions prepared from mice treated by gastrogavage with saline (control), or 5 (low), 10 (mid) or 20 (high) g/kg of cb FeD, followed by immunofluorescence staining of LC3B-II (magnification, x200). Red, LC3B-II; blue, DAPI; Merge, LC3B-II+DAPI. cb FeD, Codonopis bulleynana Forest ex Diels; LC3, LC3, microtubule-associated protein 1 light chain 3.

700712

Antibody data