Sc 32577

The HEK 293T cell line (81 (link)) was obtained from J. C. de la Torre, the A549 human cell line (82 (link)) was obtained from J. A. Melero, and the Huh7 cell line (83 (link)) was obtained from P. Gastaminza. The MDCK cell line was purchased from the ATCC. Cell culture was carried out as previously described (84 (link)). Influenza virus strains A/Wisconsin/33 (H1N1) (WSN) and A/New Caledonia/20/99 (H1N1) and a recombinant virus containing the M, HA, and NA segments of WSN in the background of A/Victoria/3/75 (H3N2) (VIC) (85 (link)) were used in these experiments. Titrations were done in MDCK cells as previously described (86 (link)). VSV was provided by R. Alfonso and titrated in BHK21 cells. Plasmids pCMVPB1, pCMVPB2, pCMVPA, and pCMVNP, expressing the influenza virus polymerase subunits and the NP, have been previously described (85 (link)). Plasmid pHHclone23, a genomic plasmid expressing a deleted NS RNA segment (clone 23) (57 (link)) in negative polarity, was provided by R. Coloma. Antiserum specific for NP was generated by immunization of rabbits with recombinant NP (57 (link)). A polyclonal antibody specific for P-PERK (SC-32577) was purchased from Santa Cruz, and a monoclonal antibody specific for actin (ab8226) was purchased from Abcam. The secondary antibodies used for Western blotting were purchased from Sigma.

Mouse liver tissues were homogenized in RIPA buffer with protease and phosphatase inhibitors (Bimake, Houston, USA). The protein concentration was determined using a BCA Protein Quantitative Assay Kit (Shenergy Biocolor Bioscience & Technology Co., Shanghai, China). Total protein was mixed with SDS loading buffer and subjected to SDS-PAGE on a 10% gel. Proteins were electrotransferred onto PVDF membranes (Millipore) and the blots were probed with the following primary antibodies overnight at 4 °C: anti-FASN (cst3180, Cell Signaling Technology, USA), anti-SREBP1C (ab3259, Abcam, USA), anti-SCD1 (ab19862, Abcam), anti-CPT1α (ab176320, Abcam), anti-MTP (sc-135994, Santa Cruz Biotechnology, USA), anti-CD36 (18836-i-ap, Proteintech, China), anti-FGF21 (ab171941, Abcam), anti-BIP (11587-1-ap, Proteintech), anti-p-IRE (ab48187, Abcam), anti-IRE (ab37073, Abcam), anti-eIF2α (cst9722, Cell Signaling Technology), anti-p-eIF2α (cst3597, Cell Signaling Technology), anti-ATF4 (10835-1-ap, Proteintech), anti-ATF6 (ab122897, Abcam), anti-p-PERK (sc-32577, Santa Cruz Biotechnology), anti-PERK (ab65142, Abcam) and anti-GAPDH (60,004–1, Proteintech). Appropriate secondary antibodies conjugated to horseradish peroxidase (Amersham) were diluted 1:5000 used. The bound primary antibodies were visualized using the Alpha Q detection system.

Active PERK and PKR and their endogenous inhibitor were immunolocalized in sequential sections from two human MTP using antibodies to phosphorylated PERK (Santa Cruz: sc-32577; 1:50), phosphorylated PKR (Santa Cruz: sc-101783; 1:50), and P58^IPK (Abcam: ac70840; 1:20). Sections were deparaffinized and rehydrated prior to antigen retrieval (1 mg/mL trypsin for 1 h at 37°C). Each subsequent step was performed at room temperature unless stated otherwise and between each incubation step, sections were washed 3 min × 5 min in 0.01 M phosphate buffered saline (PBS, pH 7.4) containing 0.001% (v/v) Tween 20 (wash buffer). All antibodies were diluted in wash buffer. Endogenous peroxidase activity was blocked with 0.3% (v/v) hydrogen peroxide for 30 min. Sections were subsequently treated with 10% normal goat serum for 1 h prior to overnight incubation (4°C) with primary antibody. Biotinylated secondary antibody was applied and incubated for 30 min detection (Vectastain Elite ABC kit, nickel enhanced diaminobenzidine, Vector Laboratories). Sections were finally dehydrated, cleared in xylene, and mounted. Slides were viewed on a Leica DMRB microscope. IgG controls were negative (Figure S2 in Supplementary Material).