Splicing xbp1

Cell lysates were prepared in RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), then were transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% non-fat dry milk for 1 h. Primary rabbit antibodies include anti-Bip (1:1000, Cell Signaling), anti-CHOP (1:1000, Cell Signaling), anti-PERK (1:1000, Cell Signaling), phospho-PERK (1:1000, Invitrogen), IRE1-α (1:1000, Cell Signaling), p-IRE1-α (1:1000; Invitrogen), splicing XBP1 (1:1000, Cell Signaling), ATF6 (1:1000, Cell Signaling). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were used and the chemiluminescent signal was detected by using electrochemiluminescence (ECL) reagents (Invitrogen).

Cell lysates were prepared in RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then were transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% nonfat dry milk for 1 h. Primary rabbit antibodies include anti-β-actin (1 : 1000, Cell Signaling), anti-CHOP (1 : 1000, Cell Signaling), anti-PERK (1 : 1000, Cell Signaling), phospho-PERK (1 : 1000, Invitrogen), IRE1-α (1 : 1000, Cell Signaling), p-IRE1-α (1 : 1000; Invitrogen), and splicing XBP1 (1 : 1000, Cell Signaling). HRP-conjugated anti-rabbit secondary antibodies were used, and the chemiluminescent signal was detected by using electrochemiluminescence (ECL) reagents.

The mice were sacrificed 48 hours after irradiation. Tumor tissues and spleen tissues were fixed in paraffin, imbedded, and cut for 4 mm sections. Tumor sections were incubated with primary antibodies, including anti-mouse CD4 (1 : 100, Cell Signaling), CD8 (1 : 100, Cell Signaling), CD11c (1 : 100, Cell Signaling), FoxP3 (1 : 100, Cell Signaling), CHOP (1 : 100, Cell Signaling), phospho-PERK (1 : 100, Invitrogen), p-IRE1-α (1 : 100; Invitrogen), and splicing XBP1 (1 : 100, Cell Signaling) antibodies, at 4°C overnight, and biotin-labeled secondary antibody for 30 min at 37°C. The final signal was developed using the 3,3′-diaminobenzidine (DAB) substrate, and the sections were observed under optical microscope. The percentage of positive cells was calculated as number of positive (brown) cells/total number of cells × 100 in 9 randomly selected fields (400×).