L929 cells (ATCC) and HeLa cells from American Type Culture Collection stably transfected with a cDNA encoding the MHC class I Kb molecule were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7.5% fetal bovine serum (FBS) at 37 °C with 9% CO2. 293T and MDCK cells from American Type Culture Collection were propagated in DMEM supplemented with 10% FBS at 37 °C with 9% CO2.
Spliceostatin A was synthesized as described (14 (link)). Cycloheximide (Sigma), anti-β-actin antibody (Sigma), MG132 (Calbiochem) were obtained from vendors. Anti-SIINFEKL C-terminal antibody (1F10.2.2) was obtained from Kenneth Rock (U Mass Med). Anti-IAV mAbs were described previously (15 (link)): anti-HA, H36-26; anti-NA, NA2-1C1; anti-M1, M2-1C6; anti-M2, O19 (16 (link)). The anti-NA rabbit polyclonal Ab used for western blotting was described (17 (link)). For flow cytometry, FITC anti-mouse H-2Kb (clone#AF6-88.5) was from BD Biosciences. MAb 25D1.16 (anti-Kb-SIINFEKL) and M2-1C6 were labeled with Alexa Fluor 647 (Life Technologies). NA2-1C1 was labeled with Pacific Blue (Life Technologies). H36-26 and O19 were labeled with Alexa Fluor 488 (Life Technologies).
Spliceostatin A was synthesized as described (14 (link)). Cycloheximide (Sigma), anti-β-actin antibody (Sigma), MG132 (Calbiochem) were obtained from vendors. Anti-SIINFEKL C-terminal antibody (1F10.2.2) was obtained from Kenneth Rock (U Mass Med). Anti-IAV mAbs were described previously (15 (link)): anti-HA, H36-26; anti-NA, NA2-1C1; anti-M1, M2-1C6; anti-M2, O19 (16 (link)). The anti-NA rabbit polyclonal Ab used for western blotting was described (17 (link)). For flow cytometry, FITC anti-mouse H-2Kb (clone#AF6-88.5) was from BD Biosciences. MAb 25D1.16 (anti-Kb-SIINFEKL) and M2-1C6 were labeled with Alexa Fluor 647 (Life Technologies). NA2-1C1 was labeled with Pacific Blue (Life Technologies). H36-26 and O19 were labeled with Alexa Fluor 488 (Life Technologies).
