Sc 9079

Unless otherwise stated, cells were lysed by shaking with acid-washed glass beads in a FastPrep FP120 device (Savant) with speed 5 for 30 s. Lysis was performed in lysis buffer A (50 mM NaCl, 50 mM Tris [pH 7.6], 0.2% Triton X-100, 0.25% NP-40, supplemented with phosphatase inhibitor cocktail 04906837001 and protease inhibitor cocktail 04693159001 [Roche]). Protein concentration was determined using a bicinchoninic acid (BCA) assay. Equal protein concentrations of each sample were loaded, and proteins were separated by SDS-PAGE and thereafter blotted onto nitrocellulose membranes.
Phosphorylation of Sty1 was detected by mouse anti-phospho-(Thr180/Tyr182)-p38 antibodies from Cell Signaling Technology (Bionordika AB, Stockholm, Sweden). HA-tagged Wis1 was detected with mouse monoclonal anti-HA (2367; Cell Signaling). The loading control was α-tubulin detected by mouse anti-α-tubulin (T5168; Sigma). Total Sty1 was detected using polyclonal rabbit anti-Hog1 (sc-9079; Santa Cruz Biotechnology) or, for MHNSTY1 (Sty1 with N-terminal Myc and His₆ tags), with mouse monoclonal anti-c-Myc (9E10, sc-40; Santa Cruz Biotechnology). The secondary antibodies were horseradish peroxidase-coupled anti-mouse A4416 and anti-rabbit A6154 (Sigma).

Ede1 constructs were detected using an anti-GFP mouse monoclonal antibody (ab291, Abcam) at 1/2000 dilution, and an anti-Hog1 rabbit polyclonal antibody (sc-9079, Santa Cruz Biotechnology) at 1/1000 dilution was used as a loading control. Donkey antimouse IRDye 680 and anti-rabbit IRDye 800 secondary antibodies (926–68072 and 926–32213 respectively, LI-COR Biosciences) were used at a 1/10,000 dilution.