Anti ncald

Brain and spinal cord were collected and lysed in RIPA buffer (Sigma, St. Louis, MO, USA) containing protease inhibitors (Complete Mini, Roche, Basel, Switzerland). The primary antibodies used: anti-beta-actin HRP-conjugated (Cat# HRP-60008, RRID:AB_2819183 Proteintech, Rosemont, IL, USA ), anti-ISL1 (mouse, Cat# 39.4D5, RRID:AB_2314683 Hybridoma Bank, Iowa City, IA, USA), anti-NCALD (rabbit, Cat# 12925-1-AP, RRID:AB_2149410 Proteintech, Rosemont, IL, USA), anti-SMN (mouse, Cat# 610646, RRID:AB_397973 BD Biosciences, San Jose, CA, USA). Signal was detected with rabbit-HRP-conjugated secondary antibody (Cat# 7074, RRID:AB_2099233 Cell Signaling Technology, Danvers, MA, USA) and mouse-HRP-conjugated secondary antibody (α-mouse, Cat# 115-035-003, RRID:AB_10015289 Jackson ImmunoResearch Labs, West Grove, PA, USA) and the chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Forty-eight hours post-transfection, cells were lysed using a lysis buffer containing the mammalian protein extraction reagent RIPA (Beyotime China), a protease inhibitor cocktail (Roche, Basel, Switzerland) and PMSF (Roche). Cells' protein lysates that contained 50 μgprotein were electrophoresed on 10% SDS-PAGE and transferred onto 0.22 mm NC membranes (Sigma–Aldrich), and incubated with specific antibodies. Specific bands were detected by ECL chromogenic substrate and quantified by densitometry (Quantity One software, BioRad). GAPDH antibody was used as control. Anti-CDK2, CDK4, CDK6 (1:1,000) were purchased from Cell Signaling Technology, Inc., anti-NCALD was purchased from Proteintech.