Activated polyvinylidene fluoride membranes

Proteins were isolated from cells and tissues using the following methods. Cells from cell culture were washed with phosphate-buffered saline and collected in lysis buffer and processed as previously outlined^{87 (link)}. Protein content was determined using the BioRad protein assay. Equal amounts of whole protein lysate were separated on SDS-polyacrylamide gels and transferred to activated polyvinylidene fluoride membranes (Millipore) Western blotting was done in keeping with standard protocols. Primary antibodies used were: anti-HELLS (ABD41, Millipore), anti-Cyclin D2 (sc-593, Santa Cruz), anti-Cyclin D1 (NBP2-32840, Novus Biologicals), anti-β-actin (4970S, Cell Signaling Technology), anti-β-tubulin (T4026, Sigma), and anti-cleaved caspase-3 (9661L, Cell Signaling Technology). Secondary antibodies conjugated to horseradish-peroxidase were: anti-mouse (715-035-150, Jackson Immuno Research), and anti-rabbit (31460, Pierce, Life Technologies). Blots were developed using Pierce ECL reagents, and chemiluminescence was detected by exposing membranes to GE-Amersham film. Blots were scanned into digital files and exposures were selected for densitometry. Densitometric analysis was done using ImageJ.

HUVECs and tissue samples were lysed using RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (Invitrogen, Carlsbad, CA, USA) and collected by centrifugation at 14 000 × rpm for 10 min. Equal amounts of proteins and pre-stained protein ladder (Thermo Fisher Scientific) were separated on 10% SDS-PAGE gels, transferred to methanol-activated polyvinylidene fluoride membranes with a 0.45 μm pore size (Millipore, Billerica, MA, USA), and incubated with primary antibodies overnight at 4 °C. The membranes were incubated with secondary antibodies (ProteinTech, Rosemont, Penn., USA) the next day for 1 h 20 min at room temperature. Bands were visualized using Immobilon ECL substrate (Millipore, Billerica, MA, USA), and blots were imaged with an LAS-4000 luminescent image analyser (Fujifilm USA, Valhalla, NY, USA). Protein expression was quantified using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA), normalized to the β-actin expression in each sample, and expressed as a percentage of the control. The primary antibodies used in the experiments are shown in Supplementary Table 2.