Human p62/SQSTM1 Antibody

Detection of Human p62/SQSTM1 by Simple Western^TM.

Simple Western lane view shows lysates of RT-4 human bladder carcinoma cell line, loaded at 0.2 mg/mL. A specific band was detected for p62/SQSTM1 at approximately 64 kDa (as indicated) using 20 µg/mL of Rabbit Anti-Human p62/SQSTM1 Monoclonal Antibody (Catalog # MAB80281). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western Blot Shows Human p62/SQSTM1 Specificity by Using Knockout Cell Line.

Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and p62/SQSTM1 knockout HeLa cell line (KO). PVDF membrane was probed with 1 µg/mL of Rabbit Anti-Human p62/SQSTM1 Monoclonal Antibody (Catalog # MAB80281) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). A specific band was detected for p62/SQSTM1 at approximately 60 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (2275-PC-100) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Western Blot Shows Human p62/SQSTM1 Specificity Using Knockout Cell Line.

Western blot shows lysates of U2OS human osteosarcoma cell line and p62/SQSTM1 knockout U2OS cell line (KO). Nitrocellulose membrane was probed with 0.5 µg/mL of Rabbit Anti-Human p62/SQSTM1 Monoclonal Antibody (Catalog # MAB80281) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody. A specific band was detected for p62/SQSTM1 at approximately 62 kDa (as indicated) in the parental U2OS cell line, but is not detectable in knockout U2OS cell line. The Ponceau stained transfer of the blot is shown. This experiment was conducted under reducing conditions. Image, protocol, and testing courtesy of YCharOS Inc. See ycharos.com for additional details.

Detection of p62/SQSTM1 by Western Blot

Loss of DENND6A impairs nutrient dependent lysosomal positioning and autophagic flux. A Unstarved or Earle’s Balanced Salt Solution (EBSS) starved HeLa cells were fixed, stained with LAMP1 antibody and imaged using confocal microscopy (Leica SP8). The cell periphery is outlined by a white dotted line. Scale bar = 10 µm. Yellow arrows indicate the presence of peripheral lysosomes. b Quantification of cumulative distribution of LAMP1 intensity in experiments performed in (a) (under starvation condition); mean ± SEM; two-tailed extra sum of F-squares test following nonlinear regression and curve fitting; n = 28, 29, 30 cells, corresponding to WT, DENND6A KO1 and DENND6A KO2, from 3 replicates. c Immunoblot showing LC3B-II protein levels under various conditions (unstarved; EBSS starved; and EBSS starved + Bafilomycin A1 (BafA1)) Immunoblot probed with anti-LC3B-II, anti-p62 and anti-HSC70 antibodies. d Quantification of experiment in (c); means ± SEM; two-way ANOVA (**P ≤ 0.0025; ***P ≤ 0.0005; ****P ≤ 0.0001; n = 3 from three replicates). e HeLa WT and DENND6A KOs cells from c were fixed and stained with LC3B-II antibody and DAPI. The cell periphery is outlined by a white dotted line. Scale bar = 25 µm. f Quantification of experiment in (e); means ± SEM; Kruskal-Wallis test ****P ≤ 0.0001; n = (32, 43, 37); (36, 34, 42); (34, 41, 34) cells, corresponding to WT, DENND6A KO1 and DENND6A KO2 in unstarved; EBSS; EBSS + BafA1, from 3 replicates). Image collected and cropped by CiteAb from the following open publication (https://www.nature.com/articles/s41467-024-44957-1), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of SQSTM1/p62 by Immunoprecipitation.

Immunoprecipitation was performed on cell lysate of U2OS human osteosarcoma cell line using 1.0 μg of Mouse Anti-Human SQSTM1/p62 Monoclonal Antibody (Catalog # MAB80281) pre-coupled to protein G or protein A beads. Immunoprecipitated SQSTM1/p62 was detected with a Mouse Anti-SQSTM1/p62 Monoclonal Antibody. The Ponceau stained transfers of each blot are shown. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitated. Image, protocol, and testing courtesy of YCharOS Inc. (ycharos.com).