P p38mapk t180 y182

BTZ (catalog S1013), CFZ (catalog S2853), MEL (catalog S8266), DEX (catalog S1322), and DOX (catalog S1208) were purchased from Selleck Chemicals. Recombinant human PSMB5 protein was custom-made by MedChemExpress. Recombinant human ISG20L2 protein was custom-made by Merry Bio Technology Co., Ltd.
Western blot antibodies against PSMB5 (catalog sc-393931), PSMD8 (catalog sc-514053), PSMD3 (catalog sc-393588), PSMC5 (catalog sc-390631), and eIF2a (catalog sc-133132) were purchased from Santa Cruz Biotechnology. Western blot antibodies against ISG20L2 (catalog 24639-1-AP) and GAPDH (catalog 60004-1-Ig) were purchased from Proteintech. Antibodies against ATF4 (catalog 11815), ATF6 (catalog 65880), cleaved caspase-3 (catalog 9661), p-p38MAPK (T180/Y182) (catalog 9211), p38MAPK (catalog 9212), p-eIF2α (S51) (catalog 3398), and XBP-1s (catalog 27901) were obtained from Cell Signaling Technology. Western blot antibodies against p-PERK (T982) (catalog WL05295), PERK (catalog WL03378), and CHOP (catalog WL00880) were obtained from Wanleibio.

Immunoblot and immunoprecipitation were performed according to the standard procedures (Kim et al., 2018 (link)). The following primary antibodies were used: anti-HDAC6, TLR2, TLR4, and SIRT1 (ABclonal); anti-FcεRIβ, Lyn, GATA3, T-bet, JNK1, pJNK1^T183/Y185, Tryptase, Chymase, BECN1, MyD88, TSG101, and Calnexin (Santa Cruz Biotechnology); anti-CXCL13 (R&D Systems); anti-CD163, FoxP3, TSLP, and MIP-2 (Abcam); anti-iNOS, pBECN1^S14, COX2, ERK1/2, pERK^T204, HDAC3, NFκB, AMPKα, pAMPKα^T172, IKBα, pIKBα^S32, p38MAPK, p-p38MAPK^T180/Y182, and LC3(Cell Signaling Technology). The detailed information of primary antibodies is described in Supplemental Table S2.To isolate tissue lysates, tissue was frozen in liquid nitrogen to preserve protein structure and homogenized with RIPA buffer. After lysis, vortexing and centrifugation at 10,000 X g for 15 min at 4°C were followed. Supernatant was then obtained and used as tissue lysates for immunoblot and immunoprecipitation.

HCMEC/D3 were grown on collagen I coated 35 ​mm² Petri dishes and stimulated with CCL4 for 5, 10, or 60 ​min, fixed in 4% paraformaldehyde and blocked in PBSA (0.5% with sodium azide). Nuclei of cells were visualised by the addition of 1 ​μg/mL Hoechst 33258 (bis-benzminide) or DAPI in the secondary antibody staining solution. The dishes were stored at 4 ​°C, mounted, and imaged using the Zeiss LSM 710 confocal microscope. Optionally, cells were permeabilised using ice cold acetone at −20 ​°C. Cells were then incubated with primary and secondary antibody at 37 ​°C in a humidified chamber. To reveal surface VE-cadherin, permeabilisation was performed after the first primary antibody incubation. Anti-Human VE-Cadherin Monoclonal Antibody (R&D Systems) and VE-cadherin Antibody (C-19) (Santa Cruz Biotechnology) were used at 1:200, P–P38 MAPK (T180/Y182) (Cell Signalling Technology) was used at 1:3000 and ZO-1 (Thermofisher) was used at 1:50, in 0.1% PBSA. Secondary antibodies were used at 1:300 in 0.1% PBSA.