Guinea pig anti p62 sqstm1

Cultured cells (neurons and MEFs) were washed with PBS and fixed for 15 min in 4% paraformaldehyde in PBS. After fixation, cells were washed with PBS and incubated for 1 h in blocking solution containing 10% FCS and 0.2% Triton X-100 in PBS. Cells were then incubated overnight at 4 °C in blocking solution containing primary antibody (1:50): goat anti-lamin B (catalog no. sc-6216; Santa Cruz Biotechnology); rabbit anti-LC3; guinea pig anti-p62/SQSTM1 (catalog no. GP62-C; Progen); rabbit anti-Syne-1 or rabbit anti-fibrillarin (catalog no. ab5821; Abcam). Coverslips were then washed with PBS, followed by secondary antibody staining for 1 h at room temperature at 1:500 dilution: anti-goat Alexa Fluor 555 (catalog no. ab150134; Abcam); anti-rabbit Alexa Fluor 488 (catalog no. ab150073; Abcam); or goat anti-guinea pig Alexa Fluor 647 (catalog no. ab150187; Abcam). VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (catalog no. H-1800; Vector Laboratories) was used to mount coverslips on slides and stain nuclei. Confocal images of fluorescently labeled proteins were captured using a 40× objective lens on an LSM 710 NL multi-photon confocal microscope (ZEISS). Puncta number, size and pixel intensity measurements were performed with ImageJ after background subtraction and threshold setting.

Dermal fibroblasts (0.1 × 10⁶ cells) were plated on glass coverslips pre-coated with 0.1% gelatin (Sigma-Aldrich) in PBS. At the end of the treatments described above, cells were washed twice with PBS and incubated in cold 1:1 methanol-acetone solution for 10 min at −20°C. The same fixation/permeabilization procedure was performed on 10-μm-thick cross cryosections of TA muscles. Slides were saturated with 10% goat serum in PBS for 30 min at room temperature and incubated overnight with the following antibodies: rat anti-LAMP1 (1:100; DSHB, clone 1D4B); rabbit anti-LC3B (1:150; Thermo Fisher Scientific, PA1-16930); guinea pig anti-p62/SQSTM1 (1:100; Progen, GP62-C); mouse anti-eMHC (1:25; DSHB, clone F1.652); rabbit anti-Laminin (1:800; Sigma-Aldrich, clone L9393). Slides were washed in PBS and incubated for 1 h at room temperature with the appropriate secondary antibodies (Jackson ImmunoResearch). Slides were mounted in 80% glycerol-PBS and images were taken by using a Zeiss LSM700 laser-scanning confocal microscope.

Antibodies used were rabbit anti-tankyrase 1/2 and mouse anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-podocin and mouse anti-tubulin (Sigma-Aldrich), goat anti-tankyrase 2, rabbit anti-BAX and rabbit anti-fibronectin (Abcam, Cambridge, UK), rabbit anti-CD2AP 1774 or 1764,^{4 (link), 51 (link)} mouse anti-PARP1 and rabbit anti-Poly(ADP-ribose) (Enzo Life Sciences, Farmingdale, NY, USA), mouse anti-active-β-catenin (Millipore, Billerica, MA, USA), rabbit anti-LEF1 and rabbit anti-phospho p38 MAPK (Cell Signaling Technology, Danvers, MA, USA) and mouse anti-WT1 (Upstate, Lake Placid, NY, USA), guinea pig anti-p62/SQSTM1 (Progen Biotechnik GmbH, Heidelberg, Germany) and mouse anti-Caspase-1 (Adipogen, San Diego, CA, USA).