β-Actin Antibody (C4): sc-47778

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

The PR-M(+) cells were lysed in a lysis buffer containing proteinase and phosphatase inhibitor at 4°C for 30 min and centrifuged at 10,000 r/min for 15 min. The cell lysates were then separated using 10% PAGE followed by transfer onto a polyvinylidene fluoride (PVDF) membrane. Membrane was blocked using 5% skim milk for 1 h at room temperature, followed by incubation at 4°C overnight with Tris-buffer saline with Tween (TBST)-diluted primary antibodies: anti-p62 (1:1000, ab56416, Abcam), anti-LC3B (1:10000, L7543, Sigma–Aldrich, St. Louis, MO, U.S.A.), anti-Bcl-2 (1:1000, sc-7382, Santa Cruz), anti-Beclin1 (1:500, sc-48341, Santa Cruz), anti-β-actin (1:5000, sc-47778, Santa Cruz) as an internal control, anti-PR (A-2) (1:5000, sc-398898, Santa Cruz), anti-PR-M (C262; 1:1000, sc-53943, Santa Cruz), anti-PR-B (B-30; 1:2000, sc-811, Santa Cruz) and anti-caspase-3 (1:1000, ab2302, Abcam). The membrane was then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h: goat anti-mouse IgG (1:5000, ab6789, Abcam) or anti-rabbit (1:5000, ab6721, Abcam). Finally, the membrane was washed six times with TSBT and developed using enhanced chemiluminescent (ECL), with the gray value of the protein bands quantified by ImageJ 1.48u software (Bio-Rad, Hercules, CA, U.S.A.). The experiments were repeated three times.

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Manufacturer protocol

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