Anti phospho p38 mapk thr180 tyr182 antibody

Aliquots of 50 ml SDB containing 1×10⁶ conidia/ml were shaken for 3 days at 25°C and then exposed to the osmotic stress of 1 M NaCl for 90 min, followed by collecting hyphal cells via centrifugation. The stressed cells were immediately homogenized in liquid nitrogen and the mixture was suspended in 20 mM PBS (pH 7.0) for protein extraction. The resultant suspension was centrifuged at 15,000 g for 30 min at 4°C and the supernatant was assessed for protein concentration with BCA Protein Assay Kit (KeyGen BioTECH, Nanjing, China). Subsequently, aliquots of protein extracts were separated through SDS-PAGE and transferred to polyvinyldene difluoride (PVDF) membranes (Millipore, Germany). Rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) antibody and anti-p38 MAPK antibody (Cell Signaling Technology, Boston, MA, USA) were then used to probe the respective phosphorylation and existence of Hog1 in the samples of each strain following the procedures of Western blot described previously [22] (link). Bound primary antibodies were revealed using horseradish peroxidase (HRP) conjugated anti-rabbit antibodies and a chemiluminescence detection system (ECL™ Amersham Biosciences).

Lysates were resolved by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), which was incubated with the primary antibody followed by incubation with anti-rabbit, anti-mouse, or anti-goat immunoglobulin-G conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA). Specific proteins were detected by using enhanced chemiluminescence (GE Healthcare, Backinghamshire, UK). The primary antibodies for Western blotting were as follows: anti-Notch1 antibody (Santa Cruz, Dallas, TX, USA), anti-Jagged1 antibody (Santa Cruz), anti-p53 antibody (DO-1) (Santa Cruz), anti-p21 antibody (Millipore, Billerica, MA, USA), anti-p16 antibody (BD Pharmingen, San Jose, CA, USA), anti-ID1 antibody (Santa Cruz), anti-phospho p38MAPK (Thr180/Tyr182) antibody (Cell signaling, Boston, MA, USA), anti-p38MAPK antibody (Cell signaling), anti-phospho SAPK/JNK (Thr183/Tyr185) antibody (Cell Signaling), anti-JNK1/3 antibody (Santa Cruz), anti-actin antibody (Cell signaling), anti-GAPDH antibody (Santa Cruz), anti-phosphoserine antibody (Abcam, Cambridge, UK) and anti-phosphothreonine antibody (Cell signaling). To assess the phosphorylation level of Id1, cell lysates were immunoprecipitated with FLAG M2 agarose (Sigma).