Supersignal west pico 34087 chemiluminescent substrate

Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences Whatman™). The membranes were washed in distilled water and blocked with Tris-buffered saline (TBS) containing Tween 20 (0.5% (w/v)), and supplemented with lyophilized low-fat milk (5% w/v) for 1 h at room temperature. The membranes were incubated with primary antibodies diluted in TBS-Tween containing low-fat milk (1% w/v) for 2 h at 37 °C with gently shaking. The primary antibodies used were: anti-heat shock protein 70 (HSP70; Stressgen, SPA-810); anti-annexin I (ANXA1, Santa Cruz Biotechnologies, sc11387); anti-Cluster of Differentiation 109 (CD109, Santa Cruz Biotechnology, sc98793); anti-heat shock protein A8 (HSPA8; bioss.com, bs-5117R); anti-Myosin heavy chain 9 (MYH9 (H40), Santa Cruz Biotechnology, sc-98,978). After primary antibodies incubation, the membranes were washed with TBS with 0.5% Tween 20 and incubated overnight at 4 °C under agitation with secondary antibodies. The secondary antibodies used were: horseradish peroxidase (HRP)-anti-mouse (Sigma A4416) or anti-rabbit (Sigma A6154). Blots were developed using a mixture of two chemiluminescence substrates developing kit (GE Healthcare Amersham^TH ECL Select^TH Western blotting detection Reagent RPN2235 and Supersignal West Pico #34087 Chemiluminescent Substrate Thermo Scientific).

Proteins (30 µg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman™, GE Healthcare Life Sciences, Amersham, UK). The membranes were blocked with Tris-buffered saline (TBS) containing Tween 20 (0.5% (w/v)) and supplemented with lyophilized low-fat milk (5% w/v) for 3 h at room temperature (TBS-T). The membranes were incubated with primary antibodies diluted in TBS-T containing 5% milk at 4 °C with gentle shaking overnight. The primary antibodies used were directed against CD81 (1:200 dilution; sc-166029, Santa Cruz Biotechnologies, Heidelberg, Germany), heat shock protein 70 (HSP70, 1:1000; Enzo, ADI-SPA-810), and annexin A1 (ANXA1, 1:400; Santa Cruz Biotechnologies, sc-114387). Subsequently, the membranes were washed with TBS-T and incubated at room temperature under agitation with secondary antibodies. The secondary antibodies used were Goat anti Mouse-HRP (1:5000; A4416, Merck, Saint-Quentin-Fallavier, France) for CD81 and HSP70 and Goat anti Rabbit-HRP (1:5000; A6154, Merck) for ANXA1. Blots were developed using a mixture of two chemiluminescence substrate developing kit (GE Healthcare AmershamTH ECL SelectTH Western blotting detection Reagent RPN2235 and Supersignal West Pico #34087 Chemiluminescent Substrate Thermo Scientific).