Ab216779

Intracellular protein was extracted from cells using the NucBuster Protein Extraction Kit (Merck, 77183) and quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). A total of 40 µg of cytoplasmic protein was diluted in laemmli buffer (Bio-Rad, 1610747) with beta-mercaptoethanol, denatured at 95 °C for 5 min and loaded onto Mini-PROTEAN TGX Gels (Bio-Rad, 4568084). Samples were transferred to nitrocellulose membranes using the TransBlot Tubro system (Bio-Rad, 170–4270) and protein visualised with Ponceau Stain (G-Biosciences). Membranes were blocked in 5% BSA TBS-T (5% BSA in 1× Tris-buffered saline and 0.1% Tween 20) for 1 h and incubated with primary antibodies overnight in 5% BSA TBS-T (MMP1 1:1000 ab137332, MMP2 1:1000 D4M2N Cell Signalling, B-actin 1:10,000 ab8226, Abcam). Membranes were washed with TBS-T and incubated in secondary antibodies (Dnk pAb to Rb IgG IRDye 680RD, ab216779, Goat pAb to Ms IgG IRDye 800CW, ab216772, Abcam) for 1 h at room temperature. Membranes were visualised using the Odyssey CLx system (Licor).

Proteins were extracted from single germ cells in the lysis buffer (150 mM NaCl, 5 mM EDTA pH 8.0, 50 mM Tris.Cl pH 8.0, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS) Twenty microliters of 1 mg/mL protein extract was loaded per lane on SDS polyacrylamide gels. For protein separation, homemade 10% SDS-PAGE gels were used. After separation, proteins were transferred to a PVDF Western blotting membrane (Roche) with a wet Western blotting apparatus (Labnet). Blots were probed with primary antibodies overnight at room temperature in PBS with 1% BSA. The following primary antibodies were used: rabbit anti VCL antibody (ab91459, Abcam) and mouse anti αTubulin (ab7291, Abcam, 1:1000). After 4 × 5 min of washing in PBS with 0.1% Tween 20, membranes were incubated with desired secondary Irdye-conjugated antibody diluted in PBS with 0.1% Tween 20 buffer, goat anti-rabbit Irdye 680RD (ab216779 Abcam, 1:10,000) and goat anti-mouse Irdye 800RD (ab216774 Abcam, 1:10,000), for 1 h on roller at room temperature or overnight at 4 °C. After washing in PBS, the blots were scanned with an Odyssey infrared imaging system (LI COR).