Anti-SQSTM1 / p62 antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Dilute primary Ab in blocking buffer with 0.1% saponin. - Incubate cells at 4°C overnight

Protocol tips
- Dilute primary Ab in blocking buffer with 0.1% saponin. - Incubate cells at 4°C overnight

Publication protocol

Denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting
Cells were lysed by radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Beijing, China), and total protein concentration was measured by using a bicinchoninic acid protein assay kit (Tiangen Biotech, Beijing, China). Extracted proteins were separated in sodium dodecyl sulfate– polyacrylamide gel electrophoresis and analyzed by Western blot. Antibodies used in Western blot analyses included anti- glyceraldehyde 3-phosphate dehydrogenase (ab181602, Abcam, Cambridge, MA), anti-LC3 (L7543, Sigma-Aldrich), anti-p62 (ab56416, Abcam), anti–caspase-8 (p-18, ab25901, Abcam), anti–total caspase-8 (catalog #4790, Cell Signaling Technology, Danvers, MA), and poly-adenosine diphosphate ribose polymerase (PARP; catalog #9532, Cell Signaling Technology). All antibodies were used according to manufacturers' instructions. Each Western blot was repeated at least three times. Intensities of protein bands were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD).

Immunofluorescence and microscopy
For colocalization of caspase-8, p62, and LC3, GH3 cells cultured on Laboratory-Tek-II Chamber slides (Sigma-Aldrich) were washed three times in phosphate-buffered saline (PBS), fixed for 20 minutes in 4% paraformaldehyde at room temperature, washed with PBS, and blocked with blocking buffer (5% bovine serum in PBS) for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer with 0.1% saponin and incubated with cells at 4°C overnight. After three washes with PBS, cells were incubated with secondary antibodies conjugated with Alexa 594, Alexa 488 (Invitrogen, Carlsbad, CA), or Alexa 647 (Cell Signaling) for 1 hour at room temperature. Then the nuclei were stained with 0.1 mg/mL 4′, 6-diamidino-2-phenylindole (D9542, Sigma-Aldrich) for 15 minutes at room temperature. Staining was visualized and photographed by using an LSM710 laser scanning confocal microscope with a ×63 oil immersion lens (Carl Zeiss, Oberkochen, Germany).

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