Rabbit anti p62

Manufactured by Proteintech

Sourced in United States

Rabbit anti-p62 is a primary antibody that recognizes the p62/SQSTM1 protein. p62 is an autophagy receptor that binds to ubiquitinated proteins and targets them for degradation through the autophagy pathway. The antibody can be used to detect and study the p62 protein in various applications.

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Close spelling variants of this product

Anti-p62 (38 citations) Rabbit anti-p62 antibody (3 citations) Anti-TM (2 citations) Rabbit anti-P62/SQSTM1 (2 citations)

13 protocols using rabbit anti p62

Immunohistochemical Profiling of Tumor Tissue

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The tumor tissues were fixed with 4% paraformaldehyde, dehydrated in a series of graded ethanol solutions, and embedded in paraffin for slicing into 5 μm slides. Afterwards, the slides were used for subsequent hematoxylin-eosin (H&E) or immunohistochemical staining. The antibodies used for immunohistochemistry were mouse anti-Cyclin D1 (1:100 dilution, Proteintech), rabbit anti-Cleaved Caspase 3 (1:100 dilution, Proteintech), rabbit anti-E-cadherin (1:100 dilution, Proteintech), rabbit anti-Vimentin (1:100 dilution, Proteintech), rabbit anti-Nrf2 (1:100 dilution, Proteintech), rabbit anti-p62 (1:100 dilution, Proteintech), rabbit anti-LC3 (1:100 dilution, Proteintech), rabbit anti-VEGFR2 (1:100 dilution, Affinity), rabbit anti-p-STAT3 (1:100 dilution, Proteintech), rabbit anti-c-Myc (1:100 dilution, Proteintech), and rabbit anti-PD-L1 (1:100 dilution, Cell Signaling Technology). Eventually, the images were obtained under an optical microscope (Nikon).

Xie C., Zhou X., Liang C., Li X., Ge M., Chen Y., Yin J., Zhu J, & Zhong C. (2021). Apatinib triggers autophagic and apoptotic cell death via VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling in lung cancer. Journal of Experimental & Clinical Cancer Research : CR, 40, 266.

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Immunohistochemistry of Autophagy Markers

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IHC was performed on paraffin-embedded tissues. It was conducted with rabbit anti-LC3 (1:200; Proteintech, cat.no. 14600-1-AP), rabbit anti-Bim (1:200; Proteintech, cat.no. 22037-1-AP), or rabbit anti-p62 (1:200; Proteintech, cat.no. 18420-1-AP) as the primary antibody. Antigen was removed at 95 °C for 20 min with 10 mM sodium citrate (pH 6.0). After blockading the sections with endogenous peroxidases and goat serum for 10 min, the primary antibodies were incubated overnight at 4 °C. DAB (Sangon Biotech, China) was used to observe tissue antigen. Finally, hematoxylin counterstained the specimens.

Li S., Gao L., Liu J., Guo C., Zheng J., Zhi K, & Ren W. (2022). The microRNA-10b-Bim axis promotes cancer progression through activating autophagy in oral squamous cell carcinoma. Cell Death Discovery, 8, 373.

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Antibodies and Chemical Inhibitors Protocol

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The mouse polyclonal antibody against CP204L and rabbit polyclonal antibody against B646L were prepared in our laboratory. Polyclonal rabbit anti-SNX32 (catalog no. 25763-1-AP), rabbit anti-p62 (catalog no. 18420-1-AP), and rabbit anti-RAB1B (catalog no. 17824-1-AP) were purchased from Proteintech. Rabbit anti-LC3B (catalog no. 3868s) and mouse anti-Myc (catalog no. 2276S) were purchased from Cell Signaling Technology. Monoclonal mouse anti-HA (catalog no. H3663), mouse IgG polyclonal antibody (catalog no. 12-371), rabbit IgG polyclonal antibody (catalog no. 12-370), and mouse anti-Flag (catalog no. F1804) were purchased from Sigma-Aldrich. Mouse anti-β-actin was purchased from Santa Cruz (catalog no. sc-58673). Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (catalog no. 4408s) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) (catalog no. 8889s) antibodies were purchased from Cell Signaling Technology; IPKine goat anti-mouse IgG heavy chain secondary antibody, HRP labeling (elimination of light chain interference) (catalog no. A25112) and IPKine goat anti-mouse IgG light chain secondary antibody, HRP labeling (elimination of heavy chain interference) (catalog no. A25012) were purchased from Abbkine. Dimethyl sulfoxide, 3-MA (autophagosome formation inhibitor), MG132 (proteasome inhibitor), and NH₄Cl (lysosome inhibitor) were purchased from Sigma-Aldrich.

Yang W., Li L., Zhang J., Wu J., Kang W., Wang Y., Ding H., Li D, & Zheng H. (2024). SNX32 is a host restriction factor that degrades African swine fever virus CP204L via the RAB1B-dependent autophagy pathway. Journal of Virology, 98(1), e01599-23.

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Autophagy Regulation in Cisplatin-Induced Cell Death

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Cisplatin and chloroquine were obtained from Sigma (St. Louis, MO, USA). The antibodies used in the experiments included: rabbit antibodies against LC3, Beclin-1, Bax and β-actin (Cell Signaling Technology, Beverly, MA, USA); rabbit anti-p62 (Proteintech, Chicago, USA); and rabbit anti-Bcl-2 (Abcam, Cambridge, UK).

Zhao X.G., Sun R.J., Yang X.Y., Liu D.Y., Lei D.P., Jin T, & Pan X.L. (2015). Chloroquine-Enhanced Efficacy of Cisplatin in the Treatment of Hypopharyngeal Carcinoma in Xenograft Mice. PLoS ONE, 10(4), e0126147.

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Monoclonal Antibodies for Alpha-Synuclein

Cited 1 time

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LS4-2G12 (1:5000 IHC; 1:2000 IF) and 9C10 (1:10,000) are mouse monoclonal antibodies raised to pSer129 epitope of αSyn and total synuclein (residues 2–21) respectively (Dhillon et al., 2017 (link)). Other antibodies used include: rabbit anti-p62 (1:2000, ProteinTech), mouse anti-ubiquitin (1:1000, EMD Millipore), rabbit Iba-1 (1:1000, Wako), mouse Cd68 (1:200, Invitrogen), rabbit anti-GFAP (1:1000, Dako), and rabbit anti-cd11b (1:1000, AbCam).

Sorrentino Z.A., Xia Y., Funk C., Riffe C.J., Rutherford N.J., Ceballos Diaz C., Sacino A.N., Price N.D., Golde T.E., Giasson B.I, & Chakrabarty P. (2018). Motor Neuron Loss and Neuroinflammation in a Model of α-Synuclein-induced Neurodegeneration. Neurobiology of disease, 120, 98-106.

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Proteomic Analysis of Rat Tissues

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Total protein lysates from rat tissues were extracted with 1× RIPA buffer and separated via SDS-PAGE for immunoblotting as described previously [33 (link)]. The resolved proteins were transferred onto nitrocellulose membranes and detected using the following primary antibodies: mouse anti-Ubqln2 (Abnova; Taipei, Taiwan), mouse anti-Flag (Sigma, St Louis, MI, USA), and the followed antibodies were purchased form Proteintech (Rosemont, IL, USA): rabbit anti-P62, rabbit anti-PSMA1, rabbit anti-PSMB7, rabbit anti-Rpt1, rabbit anti-Rpn1, rabbit anti-Histone 3, and mouse anti-GAPDH. Rat brains were fixed in 4% paraformaldehyde and then cryopreserved using 30% sucrose prior to cryostat sections. Tissues were sectioned transversely (20 μm) and stained using the rabbit anti-PSMB7.

Zhang W., Huang B., Gao L, & Huang C. (2021). Impaired 26S Proteasome Assembly Precedes Neuronal Loss in Mutant UBQLN2 Rats. International Journal of Molecular Sciences, 22(9), 4319.

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Renal Protein Extraction and Western Blot

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Total protein of renal tissues and glomerular mesangial cells was extracted by RIPA lysis buffer supplemented with protease inhibitors (Solarbio, China). Protein concentrations were determined using the BCA Protein Kit (Solarbio, China). All samples were boiled at 95°C for 5 min. Equal amounts of proteins were separated in 8–12% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, United States). After blocked with 5% non-fat milk for 2 h, the membranes were incubated with various primary antibodies overnight at 4°C, namely rabbit anti-LC3B (1:1,500, GeneTex, United States), rabbit anti-P62 (1:1,000, Proteintech, China), rabbit anti-PINK1 (1:1,000, Abcam, United Kingdom), mouse anti-Parkin (1:200, Santa Cruz Biotechnology, United States), rabbit anti-Drp-1 (1:1,000, Cell Signaling Technology, United States), rabbit anti-Mfn-2 (1:1,000, Cell Signaling Technology, United States). And then the membranes were washed 3 times with Tris-buffered saline containing Tween-20 for around 15 min and subsequently incubated with corresponding secondary antibodies for 2 h. After washing 3 times with TBST for around 15 min, the bands were detected by using ECL reagents (Abbkine, United States) and their density was quantified by using ImageJ software.

Yi X., Yan W., Guo T., Liu N., Wang Z., Shang J., Wei X., Cui X., Sun Y., Ren S, & Chen L. (2022). Erythropoietin Mitigates Diabetic Nephropathy by Restoring PINK1/Parkin-Mediated Mitophagy. Frontiers in Pharmacology, 13, 883057.

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Ferroptosis Regulation and Autophagy Interplay

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The following reagents were used: Fer‐1 (HY‐100579; MedChemExpress), deferoxamine mesylate (DFO; HY‐B0988; MedChemExpress), RSL3 (HY‐100218A; MedChemExpress), 3‐methyladenine (3‐MA; M9281; Sigma‐Aldrich).
The antibodies used were mouse anti‐FTH (sc‐376594; Santa Cruz), mouse anti‐GPX4 (sc‐166570; Santa Cruz), rabbit anti‐PTGS2 (12375‐1‐AP; ProteinTech), mouse anti‐ACSL4 (sc‐365230; Santa Cruz), rabbit anti‐NCOA4 (PA5‐36391; Thermo Fischer Scientific), rabbit anti‐LC3 (NB600‐1384; Novus), rabbit anti‐P62 (18420‐1‐AP; ProteinTech), mouse anti‐GAPDH (60004‐1‐Ig; ProteinTech), rabbit anti‐ferritin (ab75973; Abcam).

Yang R., Xu W., Zheng H., Zheng X., Li B., Jiang L, & Jiang S. (2020). Involvement of oxidative stress‐induced annulus fibrosus cell and nucleus pulposus cell ferroptosis in intervertebral disc degeneration pathogenesis. Journal of Cellular Physiology, 236(4), 2725-2739.

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Protein Isolation and Immunoblotting for Bladder Tissue

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For isolation of protein in bladder tissue, a commercial kit was used following its protocol (Beyotime). The primary antibodies were as follows (dilution 1 : 1000): rabbit anti-Pink1 (#23274-1-AP, Proteintech), rabbit anti-Prkn (#14060-1-AP, Proteintech), rabbit anti-LC3 (#14600-1-AP, Proteintech), rabbit anti- P62 (#18420-1-AP, Proteintech), mouse anti-Phospho-AMPKα (Thr12) (#50081, Cell Signaling), rabbit anti-AMPKα (#2532, Cell Signaling), rabbit anti-Phospho-Ulk1(Ser555) (#5869, Cell Signaling), and rabbit anti-Ulk1 (#8054, Cell Signaling). Secondary antibodies were anti-mouse IgG and anti-rabbit IgG (Jackson Laboratory, West Grove, PA, USA). All blots were repeated three times using tissue lysates merged from six rats' samples in each group.

Zhu X., Tao F., Zhang L., Yu Y., Xu Y., Yang G., Wei Z., Cheng Y., Yin X., Zhang X., Wei W, & Wang A. (2022). Astragaloside IV Protects Detrusor from Partial Bladder Outlet Obstruction-Induced Oxidative Stress by Activating Mitophagy through AMPK-ULK1 Pathway. Oxidative Medicine and Cellular Longevity, 2022, 5757367.

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Autophagy and Apoptosis Evaluation

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Chloroquine (CQ) and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-P62, mouse antiβ-actin, and horseradish peroxidase (HRP) goat anti-rabbit IgG were purchased from Proteintech (Chicago, IL, USA). Fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco (New York, NY, USA). Further, arecoline was purchased from Abcam (Cambridge, MA, USA). Rabbit anti-LC3 and rabbit anti-cysteine-aspartic acid protease (Caspase)-3 were purchased from Cell Signal Technology (Danvers, MA, USA).

Zhu B., Jiang Q., Que G., Dai Z, & Wu Y. (2020). Role of autophagy and apoptosis in atrophic epithelium in oral submucous fibrosis. Journal of oral science, 62(2).

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