Protease and phosphatase 3 inhibitor cocktail

Protein lysates were collected from primary human and murine spinal cord astrocytes in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) supplemented with a protease and phosphatase-3 inhibitor cocktail (Sigma-Aldrich), then 20 μg of protein was resolved on a 4–12% Tris gel and transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo system (Bio-Rad) according to standard protocols. Membranes were incubated overnight at 4 °C in Tris-buffered saline, 0.1% Tween^® 20 (TBST), and 5% powdered milk. Membranes were blotted with anti-human PD-L1 (Invitrogen; 14-9969-82), anti-mouse PD-L1 (BioLegend; 135202), and anti-β-actin (ThermoFisher Scientific; MA5-15739) antibodies, washed with TBST 3 times, and then incubated with HRP-conjugated secondary antibodies (ThermoFisher Scientific) for 1 h at room temperature. Membranes were washed with TBST 3 times and imaged using the ChemiDoc MP imaging system (Bio-Rad) after activation with ECL substrate solution.

Human brain stem and spinal cord astrocytes (ScienCell) were seeded in six‐well plates until confluent and treated with media alone or recombinant human cytokine for 24 hr. Protein lysate (20 μg) was isolated using RIPA buffer supplemented with a protease and phosphatase‐3 inhibitor cocktail (Sigma‐Aldrich). Lysates were resolved on a 4–12% Tris gel and transferred onto a PVDF transfer membrane (Invitrogen) using an iBlot2 system according to standard protocols. Blots were probed with polyclonal rabbit anti‐VCAM‐1 or ‐CXCR7 (ThermoFisher) and monoclonal mouse anti‐β‐actin (ThermoFisher) antibodies, followed by incubation with appropriate HRP‐conjugated secondary antibodies (ThermoFisher). Blots were imaged using a BioRad ChemiDoc MP imaging system.