α ha 16b12

Proteins were separated by SDS–PAGE (7.5% gels) followed by immunoblot analysis using mouse monoclonal α-Myc antibody (9E10; Santa Cruz), α-HA (16B12; Covance), α-GST (B-14; Santa Cruz), α-tetra His (Qiagen), or goat polyclonal α-Hog1 (yC-20; Santa Cruz) at a dilution of 1:10,000. Rabbit polyclonal α-phospho-p38 (T180/Y182, Cell Signaling) was used at a dilution of 1:2000 to detect phosphorylated Hog1. Secondary goat anti-mouse (Amersham) and donkey anti-goat (Santa Cruz) antibodies were used at a dilution of 1:10,000. Secondary donkey anti-rabbit (Amersham) was used at a dilution of 1:2000. All results involving immunoblot analyses were replicated at least once, and representative blots are shown.

For direct immunoblot experiments, cell lysates were prepared using the rapid boiling method (Kushnirov, 2000 (link)). Proteins were separated by SDS–PAGE (10% or 4–20% gradient gels, BioRad) followed by immunoblot analyses. Mouse monoclonal α-HA (16B12; Covance), α-GFP (Roche), α-carboxypeptidase Y (CPY) or goat polyclonal α-Mpk1 (yC-20; Santa Cruz) were used at a dilution of 1:10,000. Mouse monoclonal α-ubiquitin (P4D1; Cell Signaling), rabbit polyclonal α-phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling), and rabbit polyclonal α-Rad53 (abcam) were used at a dilution of 1:2000. Secondary goat anti-mouse (Jackson ImmunoResearch), donkey anti-rabbit (GE Healthcare), and donkey anti-goat (Santa Cruz) antibodies were used at a dilution of 1:10,000.