Hpa050918

HeLa cells were dissociated with trypsin and collected. After washing and centrifuge, 100 µL RIPA lysis buffer (Millipore 20–188) supplemented with protease inhibitor cocktail (Halt, 78429) was added and samples were incubated for 1 h on ice. After vortex, cell lysates were centrifuged at 21300 × g for 15 min at 4 °C. The supernatant was used for SDS-PAGE on 4–12% Bis-Tris gels (Invitrogen, NP0335) with Tris-buffered saline with 0.1% Tween-20 (TBS-T) at 150 V. Blotting was performed by iBlot (invitrogen) with nitrocellulose membranes (Invitrogen, IB301002) accordingly to the manufacturer’s protocol. Afterwards, membranes were incubated for 2 h in 5% milk in TBS-T. Primary antibodies [anti-CLCa/b (Millipore, AB9884, 1:5000), anti-CLCa (Sigma, HPA050918, 1:1000), or anti-CLCb (Abnova, H00001212-M01, 1:500)] were diluted in 5% milk/TBS-T and applied on the membranes overnight. After 5 times 5 min washing with TBS-T, secondary antibodies [anti-mouse IgG-HRP (Jackson ImmunoResearch Labs, 115-035-174, 1:2000) or anti-rabbit IgG-HRP (Jackson ImmunoResearch Labs, 211-032-171, 1:2000)] were diluted in 5% milk/TBS-T and applied on the membranes for 1 h. After washing with TBS-T, the membrane was imaged with ChemiDoc system (Bio-Rad) with ECL solution (cytiva, RPN2232). Then after washing, the membrane was stained with anti-βactin-HRP (Cell Signaling, 5125 S, 1:2000) and imaged.