Sqstm1 p62

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom, Canada

SQSTM1/p62 is a protein involved in the process of autophagy, which is the degradation and recycling of cellular components. It acts as a cargo receptor, recognizing and binding to ubiquitinated proteins and organelles, and directing them to the autophagosome for degradation.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

SQSTM1/p62 (5114) Anti-SQSTM1/p62 (5114) Rabbit anti-SQSTM1/p62 Anti-SQSTM1/p62 (8025, Anti-SQSTM1/p62 Rabbit monoclonal anti-SQSTM1/p62 SQSTM1/p62 (D5E2) SQSTM1/p62 antibody SQSTM1/p62 monoclonal antibody Mouse anti-p62/SQSTM1 (D-3)

169 protocols using sqstm1 p62

Comprehensive Immunoblot Antibody Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were used for immunoblot: GAPDH (cat. 5174, 1:1,000), SQSTM1/p62 (cat. 5114, 1:1,000), TFEB (cat. 4240, 1:1,000), LAMIN A/C (cat. 2023, 1:1000), AMPKα-pThr172 (cat. 2535, 1:1,000), total AMPKα (cat. 5831, 1:1,000), ACC-pS79 (cat. 1:1000), total ACC (cat. 3676, 1:1,000), S6-pS235/236 (cat. 4858, 1:1,000), total S6 (cat. 2217, 1:1,000), Na⁺, K⁺ -ATPase α1 (cat. 3010, 1:1,000), p70-S6K-pThr389 (cat. 9234, 1:1,000), total p70-S6K (cat. 2708, 1:1,000), TSC2-pThr1462 (cat. 3611, 1:1,000), total TSC2 (cat. 3635, 1:1,000), AKT-pThr308 (cat. 2965, 1:1,000), pan AKT (cat. 4691, 1:1,000), IP3R1 (cat. 3763, 1:1,000), and TFE3 (cat. 14779, 1:1000) were from Cell Signaling Technology and PPP3CB (cat. ab191374, 1:1000) was from Abcam; the following antibody was used for immunofluorescence: TFEB (cat. sc-48784, 1:100) from Santa Cruz Biotechnology and NFAT1 (cat. 5861, 1:100) and LAMP1 (cat. 9091, 1:200) from Cell Signaling Technology; the following antibody was used for immunohistochemistry: p62/SQSTM1 (cat. ab91526, 1:200) from Abcam. Cells were collected in 2× sample buffer, boiled, and sonicated for immunoblot analysis using standard western blot protocols. Uncropped blots were shown in Supplementary Fig. 7.

Wang C., Niederstrasser H., Douglas P.M., Lin R., Jaramillo J., Li Y., Oswald N.W., Zhou A., McMillan E.A., Mendiratta S., Wang Z., Zhao T., Lin Z., Luo M., Huang G., Brekken R.A., Posner B.A., MacMillan J.B., Gao J, & White M.A. (2017). Small-molecule TFEB pathway agonists that ameliorate metabolic syndrome in mice and extend C. elegans lifespan. Nature Communications, 8, 2270.

+ Open protocol

+ Expand

Multiparametric Analysis of Autophagy Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tom20 (sc‐17764, Santa Cruz Biotechnology), CERS1 (sc‐65096, Santa Cruz Biotechnology); E2F5 (E‐19, sc‐999, Santa Cruz Biotechnology), HPV16 E6 (N‐17, sc‐1584, Santa Cruz Biotechnology), HPV16 E7 (NM2, sc‐65711, Santa Cruz Biotechnology); lamin B (C‐20: sc‐6216, Santa Cruz Biotechnology); clathrin (TD.1, sc‐12734, Santa Cruz Biotechnology); ceramide (MID15B4, Enzo Life Sciences); actin (A2066, Sigma, Inc.), Drp1 (BD# 611738, Clone 22/Drp1, BD Biosciences), ACO2 (#6922, Cell Signaling Technology); MFF (ab81127, Abcam); LC3 (D11 XP^® mAb #3868, Cell Signaling Technology); ATG5 (#2630, Cell Signaling Technology); p53 (7F5, Rabbit mAb #2527, Cell Signaling Technology); pRb (4H1, Mouse mAb #9309, Cell Signaling Technology), SQSTM1/p62 (#5114, Cell Signaling Technology); SQSTM1/p62 (#5114, Cell Signaling Technology), SMCR7/MIEF2/MID49 (#PA5‐46624, Invitrogen).

Thomas R.J., Oleinik N., Panneer Selvam S., Vaena S.G., Dany M., Nganga R.N., Depalma R., Baron K.D., Kim J., Szulc Z.M, & Ogretmen B. (2017). HPV/E7 induces chemotherapy‐mediated tumor suppression by ceramide‐dependent mitophagy. EMBO Molecular Medicine, 9(8), 1030-1051.

+ Open protocol

+ Expand

Western Blotting Analysis of Autophagy and Apoptosis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blotting assay was performed to detect the expression of LC3B, p62, and GAPDH as previously described (Tian et al., 2018 (link)). The antibodies of LC3B (#2775, 1:200), p62/SQSTM1 (#5114, 1:500), cleaved caspase-3 (#9661, 1:1,000), cleaved PARP (#9544, 1:1,000), and GAPDH (#5174, 1:1,000) were purchased from the Cell Signaling Technology (CST, Danvers, MA, United States). And the intensity of bands was estimated by the Image J2X (National Institute of Mental Health, Bethesda, MD, United States). All experiments were repeated three times.

Deng Z., Li X., Shi Y., Lu Y., Yao W, & Wang J. (2020). A Novel Autophagy-Related IncRNAs Signature for Prognostic Prediction and Clinical Value in Patients With Pancreatic Cancer. Frontiers in Cell and Developmental Biology, 8, 606817.

+ Open protocol

+ Expand

Neferine-induced Autophagy and Apoptosis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Eagle’s Minimum Essential Medium (MEM), fetal bovine serum and Lipofectamine2000 were purchased from Thermo Scientific, USA. Neferine (purity ≥98%) and 3–4,5-dimethylthiazol-2-yl,2,5-diphenyltetrazoliumbromide (MTT), N-acetyl cysteine (NAC), Propidium Iodide (PI), 3-methyladenine, (3MA), 2ʹ,7ʹ-Dichlorofluorescin diacetate (DCFH2-DA) and monodansylcadaverine (MDC) was procured from Sigma Aldrich, USA. Nelubo nucifera dried seed liquid extract (Herbal Terra, USA). Antibodies against bcl-2, TCTP, caspase-3 & 9, cleaved caspase-3 & 9, PARP, pH2AX, beclin-1, LC-3, atg-4, 5 & 12, P62/SQSTM1, HRP conjugated anti-rabbit and anti-mouse antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The following reagents were obtained from the respective manufacturers as indicated in brackets: Cytochrome-c antibody (Bioss), bax antibody (ebioscience), caspase inhibitor Z-VAD-FMK (APExBIO), Annexin V-FITC (BD biosciene), caspase-3 ELISA kit (R&D system, Inc, USA). EGFP-LC3 was a gift from Karla Kirkegaard (Addgene plasmid # 11546) and all other chemicals used were of analytical grade.

Dasari S., Bakthavachalam V., Chinnapaka S., Venkatesan R., Samy A.L, & Munirathiknam G. (2020). Neferine, an alkaloid from lotus seed embryo target HeLa and SiHa cervical cancer cells via pro-oxidant anticancer mechanism. Phytotherapy research : PTR, 34(9), 2366-2384.

+ Open protocol

+ Expand

Protein Expression Analysis in Aorta and Liver

Check if the same lab product or an alternative is used in the 5 most similar protocols

In brief, aortal vessels and liver tissues were homogenized in ice-cold Tris buffer (0.01 M Tris, pH 7.4) and then lysed in extraction buffer containing 50 mmol/l Tris–HCl (pH 8.0), 150 mmol/l NaCl, 1% Nonidet-P40, 1% sodium deoxycholate, 0.1% SDS, 0.1 mmol/l dithiothreitol, 0.05 mmol/l phenylmethylsulphonyl fluoride, 0.002 mg/ml aprotinin, 0.002 mg/ml leupeptin, and 1 mmol/l NaVO₃ [24 (link)]. Proteins of aortal vessels and liver tissues were separated by electrophoresis in a 10% or 12% SDS-polyacrylamide gel, and then were transferred onto a PVDF membrane (0.22 μm/0.45 μm, Bio-Rad). Blots were first incubated with a monoclonal antibody against β-actin (1:10000, Sigma-Aldrich Chemical Company), anti-LC3B (1:1000, Cell Signaling Technology), p62/SQSTM1 (1:1000, Cell Signaling Technology) and PPARα (1:1000, Abcam Limited, Cambridge) at 4°C for 12 h. After primary antibody incubation, the membranes were washed three times in washing buffer for 10 min each and then incubated with the respective horseradish peroxidase-conjugated secondary antibodies (1:5000; Beyotime, Haimen, Jiangsu, China) for 1 h at room temperature. The densities of blots were determined using an ECL detection system (Syngen, Cambridge, UK) and results were analyzed by Chemidoc-Quantity-One software (Bio-Rad Laboratories).

Ding S., Jiang J., Yu P., Zhang G., Zhang G, & Liu X. (2017). Green tea polyphenol treatment attenuates atherosclerosis in high-fat diet-fed apolipoprotein E-knockout mice via alleviating dyslipidemia and up-regulating autophagy. PLoS ONE, 12(8), e0181666.

+ Open protocol

+ Expand

Western Blot Analysis of Cell Signaling Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein lysates were prepared with 1X Laemlli Buffer followed by 15s pulse sonication. Lysates were normalized for total protein content using BCA assay reagent (Pierce-Thermo). Proteins were separated by SDS-PAGE followed by transfer to PVDF membranes. Enhanced chemiluminescence (ECL) with West-Pico or West-Dura reagents (Pierce-Thermo). Syngene G-Box XT4 system and GeneSys software was used for ECL imaging. The following antibodies were used for Western blotting: Cleaved-PARP (Cell signaling, 1:1000), Zeb1 (Santa Cruz, 1:1000), E-cadherin (BD Biosciences, 1:1000), vimentin, phospho-S6, t-S6, Beclin 1, Atg16L1, p62/Sqstm1, LC3-I/II, p65/NFkB1, NBR1 (Cell Signaling, 1:1000) and GAPDH (Santa Cruz, 1:5000). All antibodies were diluted in 2% BSA/TBS-T solution.

Mehta A.K., Hua K., Whipple W., Nguyen M.T., Liu C.T., Haybaeck J., Weidhaas J., Settleman J, & Singh A. (2017). Regulation of autophagy, NF-κB signaling and cell viability by miR-124 in KRAS mutant mesenchymal-like NSCLC cells. Science signaling, 10(496), eaam6291.

+ Open protocol

+ Expand

Cell Lysis and Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed using NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.5% Nonidet P-40, 50 mM β-glycerophosphate, 10 mM NaF and 1 mM Na₃VO₄) with protease inhibitor (Millipore, 535140) on ice for 10 min. After centrifugation at 12,000 ×g for 5 min, the supernatant was saved as a crude cell extract. The crude cell extracts were boiled in the Laemmli buffer and then loaded onto a SDS-polyacrylamide gel [68 (link)]. The antibodies used for Western blotting are as follows: PARP-1 (Santa Cruz Biotechnology, sc-7150), p62/SQSTM1 (Cell signaling, 5114), LC3 (MBL International, PM036), Cyclin A (Santa Cruz Biotechnology, sc-751), Cyclin B1 (Santa Cruz Biotechnology, sc-752), Cyclin D1 (Santa Cruz Biotechnology, sc-753), p53 (Santa Cruz Biotechnology, sc-126), p21 (EMD Millipore, OP64), Aurora A-pT288 (Cell Signaling, 3079), Aurora A (Cell Signaling, 4718), Aurora A-pT288/Aurora B-pT232/Aurora C-pT198 (Cell Signaling, 2914), Aurora B (Cell Signaling, 3094), PLK1-pT210 (Santa Cruz Biotechnology, sc-135706), PLK1 (Cell Signaling, 4513), H3-pS10 (Upstate, 06-570), β-actin (Cell Signaling, 4970), and GAPDH (Santa Cruz Biotechnology, sc-25778).

Min Y.H., Kim W, & Kim J.E. (2016). The Aurora kinase A inhibitor TC-A2317 disrupts mitotic progression and inhibits cancer cell proliferation. Oncotarget, 7(51), 84718-84735.

+ Open protocol

+ Expand

Western Blot Analysis of Cellular Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells and tissues were lysed in RIPA lysis buffer (Beyotime) with 1 mM PMSF. Equal amount of protein was separated by SDS-PAGE and transferred to NC membrane. The membranes were washed, blocked and incubated with specific primary anti-human antibodies against CYP2E1, p62/SQSTM1 (Cell Signaling Technology, Inc., Danvers, MA, USA), LC3 (Novus Biologicals, Littleton, CO), Nrf2, HO-1, GCLC and β-actin (Abcam, Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Hangzhou HuaAn Biotech). Signals were visualized by chemiluminescent detection (Beyotime).

Yu Y., Tian Z.Q., Liang L., Yang X., Sheng D.D., Zeng J.X., Li X.Y., Shi R.Y., Han Z.P, & Wei L.X. (2019). Babao Dan attenuates acute ethanol-induced liver injury via Nrf2 activation and autophagy. Cell & Bioscience, 9, 80.

+ Open protocol

+ Expand

Protein Lysate Preparation and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein lysates were prepared using RIPA B lysis buffer (150 mM NaCl, 20 mM sodium phosphate buffer, 5 mM ethylenediaminetetraacetic acid, and 1% Triton X-100, pH 7.4) along with 1 tablet of protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN). For in vivo samples, tumor sections were cut and homogenized by magnetic beads in RIPA A lysis buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 150mM NaCl, 5mM EDTA, 5mM sodium pyrophosphate, 20mM sodium phosphate buffer, pH 7.4) along with 1 tablet of protease and phosphatase inhibitor cocktail). Total protein lysates (15 or 30 μg) were loaded onto an 8% or 12% polyacrylamide gel, which were then transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% milk in tris-buffered saline with Tween-20. Membranes were probed with specific primary antibodies: c-Met (C12), c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA), Axl, cleaved caspase 3, LC3B, p62/SQSTM1, Beclin-1, ATG5 and ATG7 (Cell Signaling, Danvers, MA), GAPDH (Millipore, Temacula, CA), and horseradish peroxidase-conjugated secondary goat anti-mouse or goat ant-rabbit antibody (Bio-Rad, Hercules, CA).

Gaur S., Wen Y., Song J.H., Parikh N.U., Mangala L.S., Blessing A.M., Ivan C., Wu S.Y., Varkaris A., Shi Y., Lopez-Berestein G., Frigo D.E., Sood A.K, & Gallick G.E. (2015). Chitosan nanoparticle-mediated delivery of miRNA-34a decreases prostate tumor growth in the bone and its expression induces non-canonical autophagy. Oncotarget, 6(30), 29161-29177.

+ Open protocol

+ Expand

Western Blot Analysis of Cell Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibodies for CyclinD1, cleaved caspase3, cleaved PARP, Beclin, p62/SQSTM1, LC3α, and β-actin were purchased from Cell Signaling Technologies (Beverly, MA, USA). Whole cell lysate was prepared using radio immunoprecipitation assay (RIPA) lysis buffer in the presence of protease inhibitors. Total cell lysates were separated using 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes, and then detected using various primary antibodies. The antibody-antigen complexes were detected using the Chemiluminescent Horseradish Peroxidase (HRP) Substrate (Millipore, MA, USA).

Liu W., Wang X., Sun J., Yang Y., Li W, & Song J. (2017). Parthenolide suppresses pancreatic cell growth by autophagy-mediated apoptosis. OncoTargets and therapy, 10, 453-461.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!