Ab59459

Liver tissues were fixed in 10% formalin and processed for hematoxylin–eosin (H&E) staining, Oil Red O staining and Sirius Red staining followed the standard procedure by Microworld Biotech, Co., Ltd. (Nanjing, China). Additionally, CD68 (ab283654, Abcam, Cambridge, UK), F4/80 (ab6640, Abcam), CD206 (ab64693, Abcam), iNOS (ab178945, Abcam), and HSP90 (ab59459, Abcam) anti-bodies were used for immunofluorescent staining. All the staining data were captured by fluorescence microscope (Nikon Ni-U, Shanghai, China).

Radio‐Immunoprecipitation assay (RIPA) (containing phenylmethylsulfonyl fluoride) lysis was used for extraction of total protein from tissues or cells. After 30 min of incubation on ice and 10 min of centrifugation (8000g, 4°C), the supernatant was collected for protein concentration detection with a bicinchoninic acid kit. Next, 50 μg protein was dissolved in 2× sodium dodecyl sulfate (SDS) loading buffer and boiled at 100°C for 5 min, followed by SDS‐polyacrylamide gel electrophoresis. Then the protein was transferred onto a polyvinylidene fluoride membrane, after which the membrane was blocked in 5% skim milk for an hour at room temperature. Afterward, the membrane was incubated at 4°C overnight with primary antibodies from Abcam against STIP1 (ab126753, 1:10,000), HSP90 (ab59459, 1:500), Cx43 (ab11370, 1:2000), and β‐actin (ab8226, 1:2000). After washing, horseradish peroxidase (HRP) labeled goat anti‐rabbit immunoglobulin G (IgG) (1:5000, Beijing ComWin Biotechnology Co., Ltd.) was appended to the membrane for 2 h of incubation, followed by treatment with electrochemical luminescence solution for color development. Finally, the protein bands were scanned and analyzed on a gel imager, and gray quantification of the western blot images was analyzed by the software Image Pro Plus 6.0 (Media Cybernetics). Each experiment was repeated three times.

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and antibiotics were from HyClone (Logan, UT, USA). Transwell inserts with an 8 μm polycarbonate membrane and cell culture plastic labware were purchased from Corning Inc. (New York, NY, USA). Collagen IV was a product of Trevigen Inc. (Gaithersburg, MD, USA). Antibodies directed to Hsp90α (ab59459) and Hsp90β (ab53497) were purchased from Abcam (Cambridge, UK). Secondary Alexa 488-labeled conjugates were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

Whole-cell lysate was harvested in boiling lysis buffer (25 mM Tris·HCl pH 7.6, 1% SDS, 1 mM EDTA, 1 mM EGTA) and 50 µg of total protein extract was loaded into an 8% acrylamide gel. The antibodies used were designed against ESRP1(Sigma-Aldrich, Milan, Italy) [55 (link)], ESRP2 (abcam ab113486), GAPDH (Santa Cruz Biotechnology, sc-32233, Dallas, TX, USA), and HSP90 (abcam ab59459).

Twenty‐four‐well plates were covered with 5% Poly‐d‐Lysine (300–400 μL per well), and H9C2 cells were inoculated on the plates at a density of 3 × 10⁴ cells/mL. H9C2 cells were fixed with 4% paraformaldehyde, blocked with 10% NGS, permeabilized with 0.1% TritonX100, and incubated overnight with rabbit anti‐rat Cx43 (ab235282, Abcam) plus mouse anti‐rat HSP70 (ab2787, Abcam) or anti‐Cx43 plus mouse anti‐rat HSP90 (ab59459, Abcam). Later, the cells were incubated (in the dark, 60 min) with goat anti‐rabbit IgG Alexa Fluor® 488 (ab150077, Abcam) and goat anti‐mouse IgG Alexa Fluor® 647 (ab150115, Abcam), stained by 4′,6‐diamidino‐2‐phenylindole, and then sealed. Finally, the slides were observed under an Olympus FV‐1000 confocal laser microscope.

Monoclonal antibody (MAb) against PRRSV nucleocapsid (N) was prepared in our laboratory as described previously (51 (link), 54 (link)). Mouse or rabbit MAbs against FLAG, HA, and β-actin were purchased from MBL and used for IP and Western blot assay. Mouse MAb against HIF-1α (ab16066), which was used for IP and Western blot assay, and rabbit anti-heat shock protein 90 (HSP90) (ab59459), anti-lamin A (ab26300), and anti-FIH-1 (ab233141) antibodies, which were used for Western blot assay, were purchased from Abcam. Rabbit polyclonal antibody against Ub (#3936), which was used for Western blot assay, was purchased from Cell Signaling Technology. CoCl₂ was purchased from Sigma-Aldrich (St. Louis, MO, USA).

For immunofluorescence, antibodies against α-tubulin (Sigma, T6199), cyclin B1 (Santa Cruz, sc-70898), Bub1 (Abcam, ab900), Mad2 (Santa Cruz, sc-28261), Hec1 (Abcam, ab3613) and SMC2 (Abcam, ab10412) and human CREST autoimmune serum (Antibodies, Inc., 15-235-0001) were used. For Western blot analysis, antibodies against Rad21 (Bethyl, A300-080A), SA2 (Bethyl, A300-581A), Smc1α (Bethyl, A300-055A), Smc3 (Bethyl, A300-060A), Mau2 (Abcam, ab46906), Sgo1 (Santa Cruz, sc-54329), Plk1 (Santa Cruz, sc-53418) and Hsp90 (Abcam, ab59459) were utilized. The anti-NudCL2 antibody was generated as described previously [19 (link)]. An antibody against actin (Sigma, T1978) was used as the loading control. The secondary antibodies for immunofluorescence analyses were Alexa Fluor 488-conjugated anti-rabbit or anti-mouse IgG and Alexa Fluor 568-conjugated anti-human IgG (Invitrogen). Goat anti-mouse or anti-rabbit secondary antibodies (LI-COR) conjugated with either Alexa Fluor 680 or IRDye 800 were used for Western blot analysis.