Human

Protein was extracted from dissected frozen tissue using RIPA buffer on ice. Protein concentration was estimated using a modified Bradford assay. Western blotting was performed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) as described previously⁹⁹. Briefly, protein lysates were separated by precast 4–20% Bis-Tris gradient gels (GenScript), followed by transferring onto PVDF membrane (Millipore). After 1 h blocking in blocking buffer (5% milk, 0.1% TBS-Tween) at room temperature, membranes were incubated overnight at 4 °C with primary antibodies. Antibodies for the following antigens were used: MAG (Proteintech cat#14386-1-AP - 1:1000-3000), MOG (Proteintech #12690-1-AP - 1:500-1000), PRKCE (Invitrogen PA5-83725 – 1:1000), p-PKCε (ser729) (Millipore 06-821-1; 1:1000), MBP (Cell signal #78896S, 1:1000), SGK1 (abcam - ab59337 1:1000), TPK1 (Fisherscientific cat# 50-172-6732 1:500), GAPDH (Proteintech 60004-1-Ig 1:1000), Actin (Proteintech 66009-1-Ig; 1:5000), Anti-mouse and anti-rabbit Peroxidase-AffiniPure Donkey IgG (H + L) (Jackson ImmunoResearch Labs Cat# 715-035-151 and 711-035-152). Detection was using enhanced chemiluminescence (cat# 1705061 or 1705062) on a Bio-Rad ChemiDoc™ Touch Imaging System. Band areas were normalized to Actin and/or GAPDH. Statistical comparisons were conducted using unpaired two-tailed t-test or Mann–Whitney test as appropriate. TPK1 was analyzed separately using similar methods as described in the mouse section and using only striatal tissue lysates. Boxplots were generated using ggplot2 (3.3.5).

Western blot analysis of OPC cultures was performed as outlined above with the following modifications. The following antibodies were used: rabbit anti-PRC-epsilon (Invitrogen PA5-83725, 1:1000), mouse anti-OLIG2 (Millipore, MABN50, 1:1000), mouse anti-CNPase (Biolegend, SMI-91, 1:5000), rabbit anti-MOG (Thermo, PA5-19602, 1:1000) and mouse anti-αTUBULIN (Calbiochem, CP06, 1:2500). Detection of target proteins was done by measuring chemiluminescence signal using ECL™ Prime Western Blotting Detection Reagent (Sigma, GERPN2232) on a ChemiDoc Imaging System (Bio-Rad). Image J was used to quantify the protein bands and αTUBULIN was used as loading control.