Cd protein assay

Membrane proteins were prepared at 4 °C on ice as previously described [16 (link)]. Briefly, 150 μL of packed RBCs was diluted into 1.5 mL of cold hemolysis buffer (HB) (5 mM disodium phosphate, 1 mM EDTA, pH 8), containing a protease and a phosphatase inhibitor cocktail, then washed up to 4 more times in the same buffer (until membranes became white) in a refrigerated Eppendorf microfuge at  25,000× g. The samples were stored frozen at −20 °C until use. The membrane protein content was quantified using the CD Protein Assay (Bio-Rad).

Cellular extracts were prepared as previously described [5 (link)]. Protein concentrations in supernatants were determined using CD Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). We performed Ponceau S staining to confirm correct protein transfer. Western blots were performed with 20–30 μg of total protein extract, using antibodies against: E2F1 (1:400, sc-256 Santa Cruz, CA, USA), E2F2 (1:400, sc-633 Santa Cruz), TK1 (1:1000, A5-29686 Invitrogen), DCK (1:400, sc-393099 Santa Cruz), TS (1:400, sc-3930945 Santa Cruz), P-CHK1 Ser345 (1:1000, 2348 Cell Signaling, Danvers, MA, USA), CHK1 (1:400, sc-7898 Santa Cruz), P-RPA Ser4/8 (1:1000, A300-245A Bethyl, Waltham, MA, USA), RPA (1:1000, ab2175 Abcam, Cambridge, UK), Phospho-CDK1 Tyr15 (1:1000, 9111 Cell Signaling), CDK1 (1:1000, ab18 Abcam), HSP90 (1:2000, sc-13119 Santa Cruz). Immunocomplexes were visualized with horseradish peroxidase-conjugated anti-mouse (1:4000, sc-3697 Santa Cruz) or anti-rabbit (1:4000, sc-2030 Santa Cruz) IgG antibodies, followed by chemiluminescence detection (ECL, Amersham, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) with a ChemiDoc camera (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry-based quantification was performed using Fiji software. Relative optical density was calculated by dividing the densitometry of the protein of interest with the respective loading control.

Membrane proteins were prepared at 4°C on ice as previously described [9 (link)]. Briefly, 150 μL of packed RBCs was diluted into 1.5 mL of cold hemolysis buffer (HB) (5 mM sodium phosphate, 1 mM EDTA, pH 8) containing a protease and a phosphatase inhibitor cocktail and then washed up to 4 more times in the same buffer (until membranes became white) in a refrigerated Eppendorf microfuge at 25000 g. The preparations were stored frozen at −80°C until use. Membrane protein content was quantified using the CD Protein Assay (Bio-Rad).