P mkk4

pcDNA6 MCV STco (codon optimised) was a gift from Patrick Moore (Addgene plasmid #40201; http://n2t.net/addgene:40201; RRID:Addgene_40201). pcDNA3.1-Flag-PP4C and RL-PP4C-TDN-HA were gifts from Marilyn Goudrealt (University of Toronto, Canada) and Tse-Hua Tan (National Health Research Institute, Taiwan), respectively. Primary antibodies targeting P-ERK1/2, ERK1/2 total, P-p38, p38 total, P-MK2, MK2 total, P-MSK1, MSK1 total, P-ATF2, ATF2 total, P-MKK4, MKK4 total and P-MKK3/6 were purchased from Cell Signalling Technologies and diluted 1 : 1000 in tris buffered saline (pH 7.6), containing 1% tween 20 (TBS-T) and 5% BSA, with the exception of P-ERK1/2 which was diluted 1 : 2000. GAPDH antibodies were purchased from Abcam and diluted 1 : 10 000 in TBS-T containing 5% non-fat milk. MCPyV ST was detected using 2T2 antibodies, kindly provided by Christopher Buck (NCI/CCR, Maryland, U.S.A.). Secondary antibodies conjugated to HRP were purchased from Dako (anti-mouse) and Cell Signaling Technologies (anti-rabbit) and diluted in TBS-T containing 5% non-fat milk 1 : 5000 and 1 : 2000, respectively. The p38 inhibitor SB202190 was purchased from Caltag Medsystems and used at 10 μM. SB202474 and U0126 were purchased from Cambridge Bioscience and used at 10 μM and 20 μM, respectively. All drugs were made up in DMSO at 1000× the concentration stated above.

A498 cells were plated in complete RPMI at 1.2 x 10⁶ and 2.0 x 10⁶ cells per T-75 flask for control and treated cells, respectively. After allowing cells to attach overnight, cells were treated with 0.1% DMSO or 100 nM englerin A for 3, 8, and 24 h. Cell extracts were prepared and Western Blot analysis was conducted as described previously [22 (link)]. Antibodies against MKK4, p-MKK4, eIF2α, p- eIF2α, p-JNK, and B-actin were obtained from Cell Signaling Technology (Danvers, MA) and that against JNK was obtained from Abcam (Cambridge, MA). Except for anti-JNK (diluted 1:2000), all antibodies were diluted 1:1000. Bands were visualized using SignalFire Plus ECL enhanced chemiluminescent substrate (Pierce) and exposed to HyBlot CL film (Denville Scientific). The film was developed with a Kodak film developer.

Forty-eight hours after transient transfection, cells were treated with inhibitors or DMSO vehicle control for the appropriate time before lysis with Triton X-100 lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor tablet (Roche). Primary antibodies: p-ERK1/2 (T202/Y204), p-MEK (S217/221), ERK1/2, MEK, p-MKK4 (S257/T261), p-MKK7 (S271/T275), MKK4, p-JNK (T183/Y185), JNK, MLK1, PARP, XIAP, cleaved caspase-3 (Cell Signaling Technology), MLK2, MLK3 (Epitomics), MLK4 (Bethyl), HA (Covance), Tubulin, Flag M2 (Sigma) and BRAF (Santa Cruz). Tubulin antibody was used at 1:10,000 dilution and Flag M2 at 1:5,000 dilution. All other antibodies were used at 1:1,000 dilution. Uncropped scans of the most important immunoblots are shown in Supplementary Fig. 6.

Biotin‐conjugated anti‐CD41 monoclonal antibody was from Abcam. Recombinant human Annexin V was from Enzo Life Sciences. SP600125 was from Calbiochem. The antibodies rabbit anti‐MKK4, p‐MKK4, c‐JNK and mouse anti‐p‐JNK were from Cell Signaling Technology. Phycoerythrin (PE)‐conjugated anti‐CD36 (clone CB38), PE‐conjugated CD36 isotype control (clone G155‐228), PE‐cy™5‐conjugated mouse anti‐CD41a antibody (clone HIP8) and fluorescein isothiocyanate (FITC)‐conjugated PAC‐1 were from BD Biosciences/Pharmingen. The 8‐iso‐prostaglandin‐F2α (8‐iso‐PG‐F2α) enzyme immunoassay (EIA) kit was from Cayman Chemical. The p‐selectin ELISA kit was from R&D Systems. The MV‐capture antibody AD‐1 was originally raised against human membrane‐bound liver alkaline phosphatase isolated from patients with liver cancer.¹⁰, ¹¹ The hybridoma cells were cultured in RPMI 1640 medium containing 10% FBS to produce antibody.