Anti actin antibody

For analysis of Tcb3 protein expression, 10 OD₆₀₀ units of Tcb3p-GFP–expressing cells post sterol depletion were prepared as described by Ohashi and colleagues [100 (link)]. Pellets were resuspended in SDS sample buffer and boiled for 5 min before SDS-PAGE. Protein transfer to nitrocellulose membranes and immunoblot conditions were as previously described [101 (link)]. To detect Tcb3p-GFP, immunoblots were incubated with a 1:1,000 anti-GFP antibody (ThermoFisher Scientific Inc., Waltham, MA) followed with 1:10,000 anti-rabbit-HRP secondary antibody (Bio-Rad Laboratories, Mississauga, ON). Actin was detected using 1:1,000 anti-actin antibody (Cedarlane, Burlington, ON) followed with 1:10,000 anti-mouse-HRP secondary antibody (ThermoFisher Scientific Inc.).
For analysis of Ysp2 protein expression, 10 OD₆₀₀ units of GFP-Ysp2–expressing cells were prepared and proteins extracted as above. To detect GFP-Ysp2, immunoblots were incubated with 1:2,000 anti-GFP antibody (Sigma-Aldrich Chemicals) followed by 1:10,000 anti-rabbit-HRP secondary antibody (Promega, Madison, WI). GAPDH was detected using 1:10,000 anti-GAPDH antibody (ThermoFisher Scientific Inc.) followed with 1:10,000 anti-mouse-HRP secondary antibody (Promega).

For analysis of Tcb3, Ist2 and Scs2 protein expression, cells were grown to mid log-phase at 30°C or temperature conditional mutants were shifted to the restrictive growth temperature as described above. 10 OD₆₀₀ units of Tcb3p-GFP, GFP-Ist2p or -Scs2p expressing cells were prepared as previously described [78 (link)]. For GFP-Scs2p immunoblots, pellets were resuspended in SDS sample buffer and heated for 10 min at 50°C before electrophoresis. For Tcb3p-GFP and GFP-Ist2p, pellets were resuspended in SDS sample buffer containing 8 M urea, and then heated for 10 min at 50°C. Protein transfer to nitrocellulose membranes and immunoblot conditions were as previously described [79 (link)]. To detect Tcb3p-GFP, GFP-Ist2p or -Scs2p, immunoblots were incubated with a 1:1000 anti-GFP antibody (ThermoFisher Scientific Inc., Waltham, MA) followed with 1:10,000 anti-rabbit-HRP secondary antibody (Bio-Rad Laboratories, Mississauga, ON). Actin was detected using 1:1000 anti-actin antibody (Cedarlane, Burlington, ON) followed with 1:10000 anti-mouse-HRP secondary antibody (ThermoFisher Scientific Inc.).