Anti sqstm1 p62

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-SQSTM1/p62 is a primary antibody that targets the SQSTM1/p62 protein. SQSTM1/p62 is a multifunctional protein involved in various cellular processes, including autophagy, oxidative stress response, and signal transduction. This antibody can be used for the detection and analysis of SQSTM1/p62 in various applications, such as Western blotting, immunohistochemistry, and immunofluorescence.

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Anti-p62 (43 citations) P62/SQSTM1 (29 citations) Anti-SQSTM1 (7 citations) Anti-p62/SQSTM1 (sc-28359) (3 citations)

19 protocols using anti sqstm1 p62

Immunofluorescence Analysis of SUMO-2/3

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HeLa cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) at 37°C with 5% CO2. LMB was added at a 10 nM final concentration from a 10 µm stock solution in 70% methanol (Sigma-Aldrich). An equivalent amount of methanol was added to control cells. The following rabbit polyclonal antibodies were used in this study: anti-SUMO-2/3 (Abcam 3742), anti-SUMO1 (Santa Cruz sc FL-101), anti-CRM1 (Santa Cruz sc-5595), anti-Histone H3 (Abcam1791), anti-p62/SQSTM1 (Santa Cruz sc-25575), anti-Ubiquitin (DAKO Z 0458), anti-Coilin (Santa Cruz sc-32860), rabbit anti-PML.⁶⁴ (link) Monoclonal mouse anti-CRM1 (BD 611 832), anti-PML (Santa Cruz sc PG-M3) and anti-p62/SQSTM1 (BD transduction Laboratories 610832) were used single or in conjunction with rabbit antibodies for double-labeling experiments.

Souquere S., Weil D, & Pierron G. (2015). Comparative ultrastructure of CRM1-Nucleolar bodies (CNoBs), Intranucleolar bodies (INBs) and hybrid PML/p62 bodies uncovers new facets of nuclear body dynamic and diversity. Nucleus, 6(4), 326-338.

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Western Blot Analysis of Autophagy and Signaling

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Whole cell lysates (40 μg) were loaded and resolved using 12% acrylamide SDS-PAGE and transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were blotted using the following primary antibodies: rabbit anti-LC3B, anti-phospho-ERK1/2, anti-total-ERK1/2, anti-PARP, mouse anti-MyD88 (Cell Signaling Technology, Danvers, MA, USA) and anti-p62/SQSTM1 (Santa Cruz Biotechnology, Dallas, TX, USA). Detection of the proteins of interest was achieved by incubating the membranes with secondary antibodies (anti-IgG HRP-conjugated) and using a chemiluminescence kit (ECL detection system, Amersham Biosciences, UK).

Wang B., Bellot G.L., Iskandar K., Chong T.W., Goh F.Y., Tai J.J., Schwarz H., Wong S.C, & Pervaiz S. (2020). Resveratrol attenuates TLR-4 mediated inflammation and elicits therapeutic potential in models of sepsis. Scientific Reports, 10, 18837.

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Investigating Oxidative Stress and Autophagy

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High-fat diet D12490 and control diet D12450B were purchased from the American Diet-Research Company. Saturated Oil Red O dyeing solution was obtained from Beijing Solarbio Technology Co., Ltd. Malondialdehyde (MDA) kit A003-1, superoxide dismutase (SOD) kit A001-3, peroxidase (POD) kit A084-1, glutathione (GSH) test kit A006-1 were from Nanjing Jiancheng Institute of Biological Engineering.Verapamil hydrochloride (V4629) was purchased from Sigma-Aldrich of the United States. The membranes were immunoblotted with anti-LC3 (Abcam, ab19, Cambridge, UK), anti-Proliferating Cell Nuclear Antigen (PCNA) (Abcam, ab29, Cambridge, UK), anti-Beclin 1 (Abcam, ab207612, Cambridge, UK), anti-p62/SQSTM1 (Santa Cruz Biotechnology, Sc-28,359, Santa Cruz, CA, USA), anti-Atg7, anti-phosphorylated and total mTOR, anti-phosphorylated and total 4EBP1, anti-phosphorylated and total p70 S6 Kinase (Cell Signalling Technology, #2631, #4060, #9272, #2971, #2983, #9205, #2708, Beverly, MA, USA). Carbon tetrachloride (CCl4) and olive oil were from Shanghai Xinzhong Chemical Reagent Factory.

Lai J.L., Lian Y.E., Wu J.Y., Wang Y.D, & Bai Y.N. (2021). Verapamil induces autophagy to improve liver regeneration in non-alcoholic fatty liver mice. Adipocyte, 10(1), 532-545.

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Immunoblotting analysis of key signaling proteins

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Anti-phosphoMKK3/6 (1:1000, D8E9, Cell Signaling Technology, Danvers, MA, USA), anti-MKK3 (1:1000, C-19, Santa Cruz Biotechnology, Dallas, TX, USA), anti-MKK6 (1:1000, D31D1, Cell Signaling Technology), anti-phospho-p38 MAPK (1:1000, E1, Santa Cruz Biotechnology), anti-p38alpha MAPK (1:1000, 9F12, Santa Cruz Biotechnology), anti-p38beta MAPK (1:1000, F-3 Santa Cruz Biotechnology), anti-p38gamma MAPK (1:1000, E-4, Santa Cruz Biotechnology), anti-p38delta MAPK (1:1000, E-3, Santa Cruz Biotechnology), anti-p62/SQSTM1 (1:500, D-3, Santa Cruz Biotechnology), anti-LC3 (1:1000, Sigma Aldrich), anti-ERCC1 (1:1000, 8F1, Santa Cruz Biotechnology), anti-PARP (1:1000, Cell Signaling Technology) and anti-actin (1:1000, 13E5, Cell Signaling Technology) antibodies.

Stramucci L., Pranteda A., Stravato A., Amoreo C.A., Pennetti A., Diodoro M.G., Bartolazzi A., Milella M, & Bossi G. (2019). MKK3 sustains cell proliferation and survival through p38DELTA MAPK activation in colorectal cancer. Cell Death & Disease, 10(11), 842.

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Western Blot Analysis of Cell Signaling

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Western blot analysis was performed using the following primary antibodies: anti-phospho-JNK (Thr183/Tyr185) and anti-JNK1 (Cell Signaling Technology); anti-LC3 (Novus Biologicals, Littleton, CO, USA) [23 (link)]; anti-p62/SQSTM1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) [24 (link)]; anti-β-actin (Sigma-Aldrich). The immune complexes were visualized using an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA). Densitometric analysis of autoradiographic bands was performed using the NIH Image J software (National Institutes of Health, Bethesda, MD, USA).

Palumbo C., De Luca A., Rosato N., Forgione M., Rotili D, & Caccuri A.M. (2016). c-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition. Journal of Translational Medicine, 14, 37.

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Antibody Optimization for Protein Analysis

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The anti‐microtubule‐associated protein antibodies 1A/1B light chain 3B (LC3B), anti‐Erk 1/2, anti‐STAT5, anti‐Akt, anti‐ubiquitin (P4D1) and anti‐PSMB5 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti‐FLT3, anti‐p62/SQSTM1 and anti‐Beta 2 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and anti‐PSMA6 was obtained from Invitrogen (Carlsbad, CA, USA). Anti‐PSMB6 and anti‐Rpn10 were purchased from Enzo (Enzo Diagnostics, Farmingdale, NY, USA), anti‐PSMD3 was obtained from Thermo Fisher (Waltham, MA, USA) and anti‐GAPDH was obtained from Sigma‐Aldrich (St Louis, MO, USA).
Bz and chloroquine were purchased from Sigma‐Aldrich, BafA was obtained from InvivoGen (InvivoGen, San Diego, CA, USA) and VT was obtained from Sigma‐Aldrich.

Lopez‐Reyes R.G., Quinet G., Gonzalez‐Santamarta M., Larrue C., Sarry J, & Rodriguez M.S. (2020). Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3‐ITD‐driven acute myeloid leukemia. FEBS Open Bio, 11(1), 48-60.

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Immunocytochemical and Western Blot Analysis

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Immunocytochemistry and Western blot analysis were performed as we described before.²⁷, ²⁸ The following primary antibodies were used: anti‐OCT4 (1:200; Santa Cruz), anti‐SOX2 (1:200; Millipore), anti‐NANOG (1:200; R&D Systems), anti‐SSEA‐4 (1:100; Developmental Studies Hybridoma Bank), anti‐TRA‐1‐81 (1:100; Chemicon), Tuj1 anti‐tubulin beta III isoform (1:200; Millipore), anti‐SMA (1:100; DAKO); anti‐AFP (1:100; DAKO), anti‐Nestin (1:200; R&D Systems), anti‐Musashi (1:200; Millipore), anti‐Map2 (1:200; Millipore), anti‐TBR1 (1:100; Abcam), anti‐CTIP2 (1:100; Abcam), Aβ₄₂ anti‐β‐Amyloid₄₂ (1:500; Calbiochem), AT8 anti‐p‐Tau (1:1000; Thermo Fisher Scientific) and anti‐LC3B (1:500; Cell Signaling), Tau5 anti‐tau (1:1000; Thermo Fisher Scientific), anti‐Mfn1 (1:1000; Abcam), anti‐Mfn2 (1:1000; Cell Signaling), anti‐DRP1 (1:1000; Cell Signaling), anti‐Fis1 (1:1000; Santa Cruz), anti‐Ub (1:4000; Santa Cruz), anti‐LAMP2 (1:1000; Santa Cruz), anti‐Beclin1 (1:1000; Cell Signaling), p62 anti‐SQSTM1 (1:1000; Santa Cruz) and anti‐β‐actin (1:10 000; Santa Cruz).

Li L., Kim H.J., Roh J.H., Kim M., Koh W., Kim Y., Heo H., Chung J., Nakanishi M., Yoon T., Hong C.P., Seo S.W., Na D.L, & Song J. (2020). Pathological manifestation of the induced pluripotent stem cell‐derived cortical neurons from an early‐onset Alzheimer's disease patient carrying a presenilin‐1 mutation (S170F). Cell Proliferation, 53(4), e12798.

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Analysis of Mitochondrial and Autophagy Proteins in Cultured Neurons

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Cultured neurons in 6-well plates were harvested at 10 weeks of differentiation as cell pellets and were resuspended in RIPA buffer (BIOSESANG) containing protease inhibitors (ThermoFisher) and phosphatase inhibitors (ThermoFisher). They were then chilled on ice for 30 min and sonicated. The supernatants were collected after centrifugation at 13,000 rpm for 30 min to remove membrane lipids. Protein concentration was determined using a BCA Protein Assay Kit (ThermoFisher), and 25 ug of protein for each sample was electrophoresed on 8% or 12% gels. The separated samples were transferred onto a PVDF membrane and incubated with target antibodies. Protein bands were visualized by Immobilon Western (Millipore) and were detected using Chemi-Doc (Bio-Rad). The following primary antibodies were used: Tau5 anti-tau (1:1,000, ThermoFisher), AT8 anti-p-tau (1:1,000, ThermoFisher), anti-OPA1 (1:1,000, Santa Cruz), anti-Mfn1 (1:1,000, Abcam), anti-Mfn2 (1:1,000, Cell Signaling), anti-Drp1 (1:1,000, Cell Signaling), anti-Fis1 (1:1,000, Santa Cruz), anti-Ub (1:4,000, Santa Cruz), anti-LC3B (1:1,000, Cell Signaling), anti-LAMP2 (1:1,000, Santa Cruz), anti-Beclin1 (1:1,000, Cell Signaling), p62 anti-SQSTM1 (1:1,000, Santa Cruz), 4G8 anti-APP (1:500, Covance), anti-p-Drp1 (Ser637, 1:1000, Cell Signaling) and anti-β-actin (1:10000, Santa Cruz).

Li L., Roh J.H., Chang E.H., Lee Y., Lee S., Kim M., Koh W., Chang J.W., Kim H.J., Nakanishi M., Barker R.A., Na D.L, & Song J. (2018). iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia. Experimental Neurobiology, 27(5), 350-364.

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Investigating the Role of LSD1 and FOXK1 in Muscle Metabolism

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Dex and insulin (from bovine pancreas) were purchased from Sigma-Aldrich, and STZ was from Wako. An LSD1 inhibitor, T-3775440 hydrochloride, was from MedChemExpress. The primary antibodies used for western blot, co-IP, immunohistochemistry, and immunocytochemistry experiments were as follows: anti-LSD1 (Abcam, ab17721), anti-FOXK1 (Abcam, ab18196), anti-ERRγ (Abcam, ab12893), anti-MHC type I (Developmental Studies Hybridoma Bank [DHSB], BA-F8), anti-MHC type IIA (DHSB, SC-71), anti-Laminin (Sigma-Aldrich, L9393), anti-Akt (Cell Signaling Technology [CST], #9272), anti-Phospho-Akt (Ser473) (CST, #9271), anti-4E-BP1 (CST, #9454), anti-Phospho-4E-BP1 (Thr37/46) (CST, #2855), anti-SQSTM1(p62) (Santa Cruz, sc-28359), anti-GAPDH (Santa Cruz, sc-25778), anti-LC3(MBL, PM036), and anti-Sin3A (Santa Cruz, sc-994). The secondary antibodies used were as follows: anti-mouse IgG-horseradish peroxidase (GE Healthcare, NA931V), anti-rabbit IgG-horseradish peroxidase (GE Healthcare, NA934V or CST, #7074), Alexa Fluor 488 anti-Mouse IgG1 (Thermo Fisher, A21121), Alexa Fluor 350 anti-Mouse IgG2b (Thermo Fisher, A21140), and Cy3 anti-Rabbit IgG (Jackson ImmunoResearch, 711-165-152). The antibodies used for ChIP experiments were anti-tri-methylated histone H3K4 (Millipore, 07-473), anti-pan histone H3 (Abcam, ab1791), and normal rabbit IgG (Santa Cruz, sc-2027).

Araki H., Hino S., Anan K., Kuribayashi K., Etoh K., Seko D., Takase R., Kohrogi K., Hino Y., Ono Y., Araki E, & Nakao M. (2023). LSD1 defines the fiber type-selective responsiveness to environmental stress in skeletal muscle. eLife, 12, e84618.

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Protein Expression Analysis in BMECs

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Proteins from the BMECs were separated on 12% SDS-PAGE gels and transferred to PVDF membranes. The primary antibodies used in this study were: anti-MAP1LC3 (Cell Signaling Technology, USA; Cat 2775), anti-ATG5 (Novus Biologicals, USA; Cat NB110-53818), anti-SQSTM1/p62 (Santa Cruz Biotechnology Inc., Germany; Cat sc-25575), anti-GAPDH (Cell Signaling Technology; Cat 2118), anti-total mTOR (Cell Signaling Technology, Cat 2983), anti-phospho-mTOR (Cell Signaling Technology, Cat 5536), anti-phospho-4E-BP1 (Cell Signaling Technology, Cat 2855), anti-total EIF2S1 (Abcam, Cambridge Science Park, UK; Cat ab70542), anti-phospho-EIF2S1 (Abcam, Cat ab32157), anti-ATF4 (Abcam, Cat ab1371), anti-total GCN2 (Abcam, Cat ab134053), and anti-phospho-GCN2 (Abcam, Cat ab75836). The luminescent fluid was prepared according to the manufacturer’s protocol, and protein expression was detected using the alpha chemiluminescent gel imaging system FluorChem E.

Xia X., Che Y., Gao Y., Zhao S., Ao C., Yang H., Liu J., Liu G., Han W., Wang Y, & Lei L. (2016). Arginine Supplementation Recovered the IFN-γ-Mediated Decrease in Milk Protein and Fat Synthesis by Inhibiting the GCN2/eIF2α Pathway, Which Induces Autophagy in Primary Bovine Mammary Epithelial Cells. Molecules and Cells, 39(5), 410-417.

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