Anti phospho akt ser473 d9e xp rabbit mab

MDCK cells in 35 mm dishes were infected with MFPT^r virus or the WT virus at 1 PFU/cell or mock infected with PBS. At 0, 5, 10 and 15 hours post-infection, the infected cells were washed 3 times with PBS and stored at -80°C. Using cell lysis buffer (0.05 M Tris-HCl pH 7.0, 0.15 M NaCl, 1% SDS, 1% Triton X-100), the cell extracts were prepared and subjected to 10% SDS-PAGE. The resulting gels were blotted on PVDF membranes and soaked with 2% BSA in TBS for blocking treatment. Blotted proteins were immuno-reacted with rabbit anti-Akt antibody (Cell Signaling Technology, MA, USA), anti-phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling Technology), anti-cleaved caspase 3 (Cell Signaling Technology), anti-PARP-1 (Santa Cruz Biotechnology, TX, USA), anti-Actin(c-2) (Santa Cruz Biotechnology), anti-Influenza A m1 (Santa Cruz Biotechnology), or anti-Influenza A ns1 (Santa Cruz Biotechnology) antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology) were used as secondary antibodies. Bands of these proteins were detected using TMB Stabilized Substrate for HRP (Promega KK, Tokyo, Japan). Semi-quantifying the expression levels of these proteins were performed using an image analyzing software, ImageJ [12 (link)] and that of actin for data normalization.

Salicylazosulfapyridine (SASP) was supplied by Sunve Pharmaceutical Co. Ltd. (China, cat#20130117). TNBS was obtained from Sigma Chemical Co. Ltd. (USA, cat#1001616585). ELISA kits for rat IL-1β, IL-10 and TNF-α were purchased from Wuhan Biological Engineering Co. Ltd. (China, cat#U20017513 and CSB-E08855r; cat#U20017514; cat#V18017512 and CSB-E11987r). RNAiso Plus kit was from TaKaRa Technologies Co. Ltd. (China, cat#D9108A). SYBR®Premix Ex Taq™ (TliRNaseH Plus) was purchased from Invitrogen Life Technologies Co. Ltd. (USA, cat#DRR820A). Reverse Transcriptase M-MLV (RNase H-) reagent was purchased from TaKaRa Technologies Co. Ltd. (China, cat#RR036). Anti-p38 antibody was obtained from Abcam Co. Ltd. (USA, cat#ab27986);Anti-nuclear factor-κB(NF-κB) p65 (D14E12) XP® Rabbit mAb was from Cell Signaling Technology (USA, cat#8242S);Anti-phospho-AKT (Ser473, D9E) XP® Rabbit mAb was purchased from Cell Signaling Technology (USA, cat#4060S).