Cellytic mt cell lysis buffer

Extraction of HUASMC cytoplasmic and membrane proteins was performed according to a previously published method.¹² The total protein was extracted using CelLytic MT cell lysis buffer (Sigma). After the samples underwent 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and incubated with the following primary antibodies: PLC-γ1, P-PLC-γ1, IP₃R and P-IP₃R (1:1000; all from Cell Signaling, Danvers, MA, USA), PKC-α, (1:200, Life Technologies), type III procollagen (COL3A1), type I collagen (COL1A) and β-actin (1:1000, all from Santa Cruz). Following incubation at 4 °C overnight, the membrane was washed and incubated with Dye 700-fluorescein-conjugated anti-rabbit second antibody (1:4000) or IR Dye 800-fluorescein-conjugated anti-mouse second antibody (1:5000) (both from Bio-Rad) at room temperature for 1 h. After the membranes were washed with Tris-buffered saline with Tween-20, the bands were scanned using an Odyssey Image Scanning System (LI-COR Biosciences, Lincoln, NE, USA). Quantitative analysis of the bands was performed using a Quantity One System (Bio-Rad, Hercules, CA, USA).