Protein expression levels were evaluated by immunoblotting analysis as previously described [23 (link)]. Briefly, whole cell lysates (WCLs) were extracted using radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and protease/phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). The prepared WCLs were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The following primary antibodies were used: anti-cleaved caspase-3, anti-total caspase-3, anti-β-actin, anti-light chain 3 (LC3)-I/II, anti-p62, anti-Atg5, anti-Unc51 like autophagy activating kinase 1 (ULK1), anti-Beclin-1 (Abcam, Cambridge, MA, USA), anti-total mammalian target of rapamycin (mTOR), anti-phosphor-mTOR1 (p-2448S), anti-phosphor-mTOR2 (p2481S), antitotal-protein kinase B (Akt), anti-phosphor-Akt, anti-raptor, and anti-rictor (Cell Signaling Technology, Danvers, MA, USA).
Anti p62
Manufactured by Abcam
Sourced in United Kingdom, United States, China, Germany
Anti-p62 is a primary antibody that binds to the p62 (also known as SQSTM1) protein. p62 is a multifunctional protein involved in various cellular processes, including autophagy and protein degradation. This antibody can be used in techniques like Western blotting and immunohistochemistry to detect and analyze the p62 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3, by Abcam (27 mentions)
Anti beclin1, by Abcam (26 mentions)
Anti gapdh, by Abcam (18 mentions)
Anti lc3b, by Abcam (16 mentions)
Pvdf membrane, by Merck Group (16 mentions)
Anti bcl 2, by Abcam (15 mentions)
Anti β actin, by Abcam (13 mentions)
Anti bax, by Abcam (12 mentions)
Anti atg5, by Abcam (11 mentions)
Anti lc3, by Cell Signaling Technology (10 mentions)
Anti caspase 3, by Abcam (10 mentions)
Anti beclin 1, by Cell Signaling Technology (9 mentions)
Anti mtor, by Cell Signaling Technology (9 mentions)
Anti mtor, by Abcam (8 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (6 mentions)
Anti lc3b, by Cell Signaling Technology (6 mentions)
Anti gapdh, by Cell Signaling Technology (6 mentions)
Anti akt, by Abcam (6 mentions)
Anti β actin, by Proteintech (6 mentions)
Anti gapdh, by Proteintech (6 mentions)
138 protocols using anti p62
1
Protein Expression Analysis by Immunoblotting
2
Monitoring Protein Markers During Cellular Differentiation
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Protein lysates were prepared in a RIPA lysis buffer using ice and a 27 G needle with the addition of protease (Roche, 11836170001, Indianapolis, IN, USA) and phosphatase (Roche 4906837001) inhibitors. Quantification was performed using a detergent-compatible Bradford reagent (Thermo Scientific, 1863028, Waltham, MA, USA). The proteins were loaded into a 12% stain-free gel, and blocking was performed using 3% low-fat milk for 2 h. The membrane was incubated overnight at +4 °C with the following antibodies: anti-PINK1 (Abcam, ab23707, Waltham, MA, USA) 1:1000, anti-LC3 (Cell Signalling, #3868) 1:1000, anti-FUNDC1 (Novus Biologicals, NBP1-81063, Centennial, CO, USA) 1:2500, and anti-p62 1:15,000 (Abcam, ab109012). The membranes were then washed 3 times in TBST and incubated with secondary antibodies (1: 200,000) for 1 h. Subsequent detections were accomplished using the Femto SuperSignal chemiluminescent reagent (Thermo Scientific, 34095). Because of the low protein yield on D1, Western blot was only performed on D7 and D14 of differentiation. The results obtained with the Western blot were normalized to the total protein amount. All images of the Western blots can be found in the supplementary data .
3
Cisplatin-Induced Cell Viability Assay
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The reagents used in this study include the following: cisplatin (Sigma‐Aldrich, St. Louis, MO), 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) (Sigma‐Aldrich) and ViaFect™ transfection reagent (Promega, Madison, MI). Antibodies used in this study include anti‐p62 (Abcam, Cambridge, MA, USA), anti‐LC3 (Abcam), anti‐Caspase 8 (Proteintech, Chicago, IL), anti‐actin (Proteintech) and anti‐ubiquitin (Santa Cruz, CA).
4
Thapsigargin and Rapamycin Induced Cell Stress Response
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Thapsigargin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin was from Invitrogen (Carlsbad, CA, USA). DMEM and FBS were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). The Hoechst kit and Lyso-Tracker Red probe for acidic lysosome staining were from Beyotime (Haimen, Jiangsu, China). Anti-PERK, Anti-BiP, anti-p-eIF2α, anti-eIF2α, anti-cleaved-caspase 3, anti-LC3, anti-p-AKT, anti-p-p70S6K1, anti-p70S6K1, anti-p-4EBP1 and anti-4EBP1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ATF-6, anti-p62 and anti-GAPDH were from Abcam (Cambridge, UK). The CHOP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were obtained from Sigma-Aldrich with the highest purity available.
5
Investigating Apoptosis and NF-κB Signaling
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Cisplatin, trypan blue and 3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). The FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences (State of New Jersey, USA). ViaFect Transfection Reagent was purchased from Promega (Madison, MI, USA). Nuclear and cytoplasmic protein extraction kit was purchased from Beyotime (China). The following anti‐body were used: anti‐p62 (Abcam, Cambridge, MA, USA); anti‐p50, anti‐p65, anti‐β actin (Proteintech, Chicago, IL, USA); anti‐RIP1, anti‐K63‐linkage Specific Polyubiquitin (Cell Signaling Technology, Beverly, Massachusetts, USA); anti‐Lamin A/C, anti‐IκB, anti‐pIκB (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
6
Protein Extraction and Western Blot Analysis
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Nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo‐Fisher). The following primary antibodies were used for western blot analysis: anti–phosphorylated c‐Jun NH2‐terminal kinase (P‐JNK; no. 9255; Cell Signaling Technology), anti‐JNK (no. 9525; Cell Signaling Technology), anti–phosphorylated extracellular signal–regulated kinase (P‐Erk) 1/2 (no. 4376; Cell Signaling Technology), anti‐Erk1/2 (no. 5013; Cell Signaling Technology), anti‐P‐p38 (no. 9216; Cell Signaling Technology), anti‐p38 (no. 9212; Cell Signaling Technology), anti‐LC3I/II (no. 4108; Cell Signaling Technology), anti‐Nrf2 (no. 137550; Abcam), anti‐p62 (no. 109012; Abcam), and anti‐Slc7a11 (no. 37185; Abcam).
7
Immunoblotting of mouse brain homogenates
8
Histological and Immunohistochemical Analysis of Liver Autophagy
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Livers were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into four micromrter thick slices. The liver sections were deparaffinised in xylene, dehydrated in graded alcohol and washed with ddH2O. The liver sections were stained with hematoxylin and eosin according to the manufacturer’s protocols. For immunostaining, liver slices were blocked with 5% BSA and incubated overnight at 4°C with anti-ATG5 (1:200; Proteintech), anti-Beclin-1 (1:200; Proteintech), anti-TET2 (1:200; Proteintech), and anti-p62 (1:200; Abcam) after heat mediated antigen retrieval with citrate for 20 min. The slices were incubated with appropriate secondary antibodies at room temperature for 1 h, washed with sterile PBS for 15 min, stained with DAB, counterstained with hematoxylin, dehydrated in graded alcohol, rinsed with xylene, and mounted on slides. Images were acquired by an Olympus I3-TPC microscope.
9
Autophagy Regulation in Podocytes
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Rapamycin (RP) and chloroquine (CQ) were purchased from Sigma-Aldrich; Merck KGaA. Antibodies against LC3, mammalian target of rapamycin (mTOR) and p-mTOR were acquired from Cell Signaling Technology, Inc. Anti-Cx43, anti-podocin, anti-nephrin and anti-p62 antibodies were obtained from Abcam. Anti-GAPDH was purchased from CWBio.
10
Immunoblotting of Glioblastoma Proteins
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Human glioblastoma cells or tumor tissues were harvested and washed with ice-cold PBS. Then, samples were suspended in SDS sample buffer, boiled for 10 min, and centrifuged at 12,000 rpm for 15 min. Thirty micrograms of protein samples were separated using 8–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore Corporation). The PVDF membrane was blocked with 5% milk for 1 h and then incubated with a primary antibody at 4°C overnight. Subsequently, the PVDF membrane was probed with the secondary antibody and visualized by enhanced chemiluminescence (ECL) (Beyotime Institute of Biotechnology, Haimen, China). The primary antibodies were as follows: anti-G9a (1:500, Santa Cruz), anti-a-tubulin (1:2000, Sigma), anti-CDK1 (1:1000, Abcam Cambridge, MA, United States), anti-CDK2 (1:500, Santa Cruz), anti-Cyclin A2 (1:2000, Abcam), anti-Cyclin B1 (1:3000, Abcam), anti-MMP1 (1:1000, Abcam), anti-N-cadherin (1:5000, Abcam), anti-LC3B (1:1000, Cell Signaling Technologies, Danvers, MA, United States), anti-p62 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), and anti-H3K9me2 (1:2000, Abcam). Horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000), goat anti-rabbit IgG (1:20,000), and rabbit anti-goat IgG (1:10000) (KPL, Gaithersburg, Maryland, United States) were used as secondary antibodies.
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