Femto supersignal chemiluminescent reagent

Protein lysates were prepared in a RIPA lysis buffer using ice and a 27 G needle with the addition of protease (Roche, 11836170001, Indianapolis, IN, USA) and phosphatase (Roche 4906837001) inhibitors. Quantification was performed using a detergent-compatible Bradford reagent (Thermo Scientific, 1863028, Waltham, MA, USA). The proteins were loaded into a 12% stain-free gel, and blocking was performed using 3% low-fat milk for 2 h. The membrane was incubated overnight at +4 °C with the following antibodies: anti-PINK1 (Abcam, ab23707, Waltham, MA, USA) 1:1000, anti-LC3 (Cell Signalling, #3868) 1:1000, anti-FUNDC1 (Novus Biologicals, NBP1-81063, Centennial, CO, USA) 1:2500, and anti-p62 1:15,000 (Abcam, ab109012). The membranes were then washed 3 times in TBST and incubated with secondary antibodies (1: 200,000) for 1 h. Subsequent detections were accomplished using the Femto SuperSignal chemiluminescent reagent (Thermo Scientific, 34095). Because of the low protein yield on D1, Western blot was only performed on D7 and D14 of differentiation. The results obtained with the Western blot were normalized to the total protein amount. All images of the Western blots can be found in the supplementary data.

Proteins in cell lysates, obtained by RIPA lysis buffer with added protease and phosphatase inhibitors, were sonicated for 10–15 seconds with 20–30% sonicator power (Qsonica CL188, Cole-Parmer, Vernon Hills, IL, USA). Protein concentrations were quantified by using bicinchonic acid method. The proteins were loaded into a 12% stain-free gel, and the blocking was performed using a 5% low fat milk for 1 h. The membranes were incubated overnight at +4 °C with the following antibodies: anti−TUBB3 (802001, Biolegend, San Diego, CA, USA)) 1:5000, anti−MBD1 (NV100-55537, Novus Biologicus, Littleton, CO, USA) 1:1000, anti−GFAP (ab4674, Abcam, Cambridge, UK), anti−SOX2 1:5000 (#23064, Cell Signaling, Danvers, MA, USA). The membranes were then washed three times in TBST and incubated with secondary antibodies (1:200,000) for 1 h. Subsequent detections were made using Femto SuperSignal chemiluminescent reagent (34095, Thermo Fisher Scientific, Waltham, MA, USA). The results obtained with the Western blot were normalized to the total protein amount. Images of full blots with loading controls can be found in the Supplementary Data.