Cell pellets were resuspended in Laemmli sample buffer (BioRad), and Western blots were performed as described previously [45 (link)]. The membranes were incubated with the following primary antibodies: anti-lamin A/C (Thermo Fisher, 1/5000), anti-prelamin A (Santa Cruz, 1/3000), anti-53BP1 (Bethyl, 1/3000), anti-Rad51 (NBP2-32622, Novus Biological, 1/1000), anti-Lamp2 (Santa Cruz, 1/1000), anti-p62 (NovusBio, 1/3000), anti-LC3B (Sigma-Aldrich, 1/10000), and anti-β-actin (Sigma-Aldrich, 1/10000). Afterwards, the membranes were incubated with a corresponding secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories). Proteins were visualized with a chemiluminescence detection system (ECL substrate; BioRad) and signals were analyzed using the IMAGE LAB software (BioRad). Protein signals were quantified by normalizing to β-actin, as indicated.
Anti p62
Manufactured by Novus Biologicals
Sourced in United States
Anti-p62 is a laboratory reagent used for the detection and analysis of the p62 protein. p62 is a multi-functional protein involved in various cellular processes. The Anti-p62 product is designed to facilitate the study of p62 expression and function in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti flag, by Merck Group (2 mentions)
Anti lc3, by Novus Biologicals (2 mentions)
Anti lc3b, by Merck Group (1 mentions)
Anti 53bp1, by Fortis Life Sciences (1 mentions)
Anti rad51 nbp2 32622, by Novus Biologicals (1 mentions)
Anti lamp2, by Santa Cruz Biotechnology (1 mentions)
Anti lamin a c, by Thermo Fisher Scientific (1 mentions)
Anti prelamin a, by Santa Cruz Biotechnology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti lc3ii, by Proteintech (1 mentions)
Protein g, by GE Healthcare (1 mentions)
Sepharose a beads, by GE Healthcare (1 mentions)
Anti cdk1, by Cell Signaling Technology (1 mentions)
Anti cyclin b, by Santa Cruz Biotechnology (1 mentions)
11 protocols using anti p62
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Neuronal Proteins
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The proteins that were extracted from primary spinal neurons and the sciatic nerve tissues were electrophoresed on 12% SDS-polyacrylamide gel and then transferred to nitrocellulose membranes (Millipore, Carrigtwohill, Ireland), blocked with nonfat milk, and probed with primary antibodies at 4°C overnight; the membranes were washed three times and immunoblotted with secondary antibodies at room temperature for 2 hr. Finally, blots were detected by enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA), and protein bands were quantitated with ImageJ software (NIH) using β-actin as an internal control. Primary antibodies were as follows: anti-LC3-II (Proteintech, Chicago, USA), anti-p62 (Novus, Littleton, Colorado, USA), and anti-β-actin (A5441, Sigma-Aldrich Chemicals, St. Louis, MO, USA).
3
Immunoprecipitation of CDK1, p62, and Flag
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NP40 extracts (1–2 mg) were incubated with normal mouse or rabbit sera for 30 minutes and subsequently with protein G or A-sepharose beads (GE Healthcare), respectively, for 1 hour at 4 °C. After centrifugation, beads were discarded and supernatants incubated for 2 hours with anti-CDK1 (Cell Signaling Technology), anti-Flag (Sigma-Aldrich) or normal mouse (Santa Cruz Biotechnology) monoclonal antibodies or serum, or anti-p62 (Novus Biologicals) polyclonal antibody or normal rabbit (Santa Cruz Biotechnology) serum, followed by protein G or A-sepharose beads for 1 hour. Beads were washed and bound proteins were solubilized by the addition of SDS-sample buffer heated at 95 °C for 5 minutes.
4
Western Blot Analysis of Cellular Proteins
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Equal amounts (20–30 μg) of proteins were subjected to 10–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-CDK1 mouse (1:1,000) and anti-HDAC6 rabbit (1:1,000) monoclonal, and anti-K63-linkage specific polyubiquitin rabbit (1:500) polyclonal antibodies (Cell Signaling Technology, Danvers, MA, USA); anti-p62 (1:1,000), anti-LC3 (1:400) and anti-NBS1 (1:1,500) rabbit polyclonal antibodies (Novus Biologicals, Littleton, CO, USA); anti-Flag (1:5,000) and anti-α Tubulin (1:20,000) mouse monoclonal, and anti-ATG5 rabbit (1:1,000) polyclonal antibodies (Sigma-Aldrich); and anti-p53 (1:500) and anti-cyclin B (1:1,000) mouse monoclonal antibodies (Santa Cruz Biotechnology). Peroxidase-coupled donkey anti-rabbit IgG (1:10,000) and sheep anti-mouse IgG (1:10,000) were obtained from GE Healthcare (Little Chalfont, UK). Immunoreactive bands were visualized using the Enhanced Chemiluminescence Western blotting system (ECL, GE Healthcare). Quantification of protein levels was carried out using ImageJ software (Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ ).
5
Synthesis and Evaluation of Urushiol Derivatives
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Four urushiol derivatives 3-pentylcatechol (PC), 3-decylcatechol (DC), 3-pentadecylcatechol (PDC), and 3-eicosylcatechol (EC) were synthesized as reported in a previous study [3 (link)] (Figure 1A ). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tunicamycin, rapamycin, chloroquine (Cq), and 4-phenylbutyric acid (4-PBA) were purchased from Sigma-Aldrich. SP600125 (SP600) and BAPTA-AM were purchased from Calbiochem. Antibodies against mTOR, p-mTOR (Ser2448), p-S6K1 (Thr389), S6K1, p-ULK1 (Ser757), ULK1, PERK, eIF2α, p-eIF2α (Ser51), IRE1α, c-Jun, p-c-Jun (Ser63), JNK, p-JNK (Thr183/Tyr185) and LC3A/B were purchased from Cell Signaling Technology. Polyclonal rabbit anti-LC3B, p-IER1α (Ser724), and anti-p62 were purchased from Novus Biologicals and Sigma-Aldrich, respectively. Anti-β-actin, anti-HSP90, and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Polyclonal rabbit anti-p-ATG14L (Ser29) and anti-ATG14L was generated as previously described [17 (link)].
6
Apoptosis and Autophagy Pathway Analysis
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Anti-caspase 3, anti-caspase 9, anti-caspase 8, anti-PARP (Cell Signaling, Danvers, MA, United States), anti-mTOR (Spring Bioscience, Pleasanton, CA, United States), anti-phospho mTOR (Santa Cruz, Dallas, TX, United States), anti-Bcl-2, anti-Bax (Abcam, Cambridge, MA, United States), anti-p62, anti-Beclin-1, anti-light chain 3 (LC3) and anti-GAPDH (Novus Biologicals, Littleton, CO, United States).
7
Western Blot Analysis of Signaling Pathways
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At the end of the experiment, cells were detached by trypsinization and lysed in lysis buffer (100 mM Tris HCl pH 7.4, 300 mM NaCl, 0.5% NP40, protease inhibitor cocktail, phosphatase inhibitors). Upon 30 min incubation on ice, lysates were centrifuged at 14,000× g for 30 min at 4 °C and supernatants were collected. Following the determination of protein content by the Bradford assay, 50 μg of proteins were separated by SDS-PAGE and analysed by Western blotting using specific antibodies. Antibodies from Cell Signalling (Danvers, MA, USA) were used at 1:1000 dilution: anti-phospho Akt (#4060S; lot: 16), anti-phospho p38 (#4111P; lot: 10), anti-phospho SAPK/JNK (#4668P; lot: 11), anti-caspase-3 (#9664P; lot: 20), anti-phospho TSC2 (#3617; lot: 2), anti-LC3 (#2775S; lot: 5), anti-phospho p70 (#9205; lot:16), anti-p70 (#9202; lot: 14), anti-TSC2 (#3612; lot: 3). The anti-p62 was from Novus Biologicals, Littleton, CO, USA; 1:5000 dilution; #H00008878-M01; lot: FC071-2C11); anti-GAPDH (Thermo Scientific, USA; 1:10000 dilution, #PA1-988; lot: OH188198); anti β-Actin (Sigma-Aldrich; 1:1000 dilution, #A4700; lot: 036H4857). Detection was performed according to the manufacturer’s instruction, using a ChemiDoc (Biorad, Hercules, CA, USA).
8
Autophagy Modulation Assay Protocol
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3-methyladenine (3MA), bafilomycin A1 (BA), tetrakis [(2-pyridylmethyl)ethylenediamine] (TPEN), zinc chloride (Zn), ammonium chloride (NH4Cl), leupeptin, and pepstatin A were purchased from Sigma (St. Louis, MO, USA). Anti-microtubule-associated protein light chain 3 (LC3) and anti-p62 antibodies were obtained from Novus (Littleton, CO, USA) and MBL (Nagoya, Japan), respectively. Anti-β-actin and anti-beclin1 antibodies were from Cell Signaling (Beverly, MA, USA).
9
Detecting Autophagy Markers in Cells
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Protein was extracted from cells by RIPA lysis buffer (Beyotime, Haimen, China, Cat. no. P0013B) with 1 mM PMSF. Equal amounts of protein was separated by SDS-PAGE and transferred onto NC membrane. After blocking with 5% non-fat milk, the membrane was probed with anti-p62 (Novus Biologicals Inc., Littleton, CO, USA Cat. no. NBP1-48320) and anti-LC3 (Novus Biologicals Inc. Cat. no. NB100-2220H), developed with the BeyoECL Plus substrate system (Beyotime, Cat. no. P0018). Anti-GAPDH (Cell Signaling, Danvers, MA, USA, Cat. no. 2118) was used as an internal control to confirm equal protein loading.
10
Subcellular Fractionation and Western Blot
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Different cells were harvested and fractionated to produce cytosolic and nuclear lysates using the NE-PER kit (Thermo Fisher Scientific). For whole cell lysates, cells were lysed in RIPA buffer. Both protease and phosphatase inhibitors were included in the respective lysis buffer. ~47 μg of protein was separated on a 4%-15% gradient SDS-polyacrylamide gel (Bio-Rad) which were transferred to nitrocellulose membranes, stained with Ponceau S stain, washed, blocked with 5% non-fat milk, and incubated overnight at 4°C with the following primary antibodies: anti-α-Tubulin (Cell Signaling Technology (C.S.T.) #2144), anti-Lamin A/C (C.S.T. #4777), anti-SON (Abcam #121033 and LSBio LS-C803664), anti-YAP1 (C.S.T. #12395), anti-GFP (C.S.T. #2956), anti-Tau (Sigma-Aldrich, #A0024), anti-p-Tau (a gift from Dr. Peters Davies), anti-β-actin (C.S.T. #4970), anti-puromycin (BioLegend 381502), anti-ubiquitin (C.S.T. #58395), anti-LC3-I/II (C.S.T. #2775), anti-p62 (C.S.T. #23214), anti-ATF4 (C.S.T. #11815), anti-ATF6 (Novus 70B1413.1), and anti-XBP1s (BioLegend 658802). Membranes were treated with the appropriate secondary antibody conjugated to horseradish peroxidase the following day and then ECL Prime Western Blotting Detection Reagent (Cytiva) was applied. A Bio-Rad ChemiDoc MP Imaging System was used to visualize the signal, and signal intensities were determined with ImageJ 112 (link).
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