Mouse anti gapdh

Cultured FLS (passage 4) were plated at 2 × 10⁵ cells in six-well plates overnight in DMEM containing 10% FBS and synchronized for 24 h in DMEM/0.1% FBS. The cells were pretreated for 1 h with different concentrations of TRYP, TRYP-Ox, or DMSO (vehicle control) and then stimulated with 2 ng/ml IL-1β or medium for 15 min at 37°C. The cells were then washed once with ice-cold PBS and lysed with modified radioimmunoprecipitation assay buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2.5 mM MgCl₂, 1.0 mM EDTA, 20 mM β-glycerophosphate, 10 mM NaF, 1 mM Na₃VO₄, and Protease inhibitor cocktail (Roche, Indianapolis, IN)]. Protein concentration of the lysates was measured using a MicroBCA Assay kit (Pierce, Rockford, IL), and 40 μg lysate was subjected to 10% SDS-PAGE and Western blot analysis. Anti–phospho-c-Jun (Ser63) was purchased from Cell Signaling Technology (Danvers, MA), and anti-mouse GAPDH was from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunoblotting was performed according to a previously published protocol.¹³ Briefly, cell lysates were prepared in RIPA (Radioimmunoprecipitation assay) buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM disodium EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β‐glycerophosphate, 1% Triton X‐100) plus 1 mM phenylmethylsulphonyl fluoride and Halt protease/phosphatase inhibitor cocktail (Thermo Scientific). Protein concentration was measured by Bradford assay (Thermo Scientific). 20–30 μg of protein was run on precast 4–15% sodium dodecyl sulfate polyacrylamide gels (Bio‐Rad), transferred to polyvinylidene fluoride membranes (GE Water and Process Technologies), blocked in Tris‐buffered saline (pH 7.4) plus 0.1% Tween‐20 and 5% skim milk, and incubated overnight at 4°C with 1:1000 diluted antibodies against indicated proteins (Cell Signaling) plus anti‐mouse GAPDH (Santa Cruz Biotechnology) or Vinculin (Cell Signaling) as loading controls. Membranes were incubated with species‐appropriate horse radish peroxide‐conjugated secondary antibodies for 1–2 h. Proteins were detected by enhanced chemiluminescence using Western Lightening Plus reagent (Perkin Elmer). Band quantification and normalization to total protein was by ImageJ software.¹⁸ Data are shown as means of three individual blots with comparisons only made between like blots from the same gels.

Protein extraction from cells was performed using radioimmunoprecipitation assay (RIPA) buffer. Protein concentrations were determined using the BCA method (P0010S; Beyotime Institute of Biotechnology). Equal amount of protein lysates were electrophoresed by 10% SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked by TBST (NaCl 500mM, Tris 20 mM, pH 7.5) containing 5% skim milk. Protein expression in tissue samples was probed with rabbit polyclonal anti-PSMC2 (sigma, F1804) and mouse anti-GAPDH (Santa Cruz Biotechnology, CA).Mouse anti-FLAG and anti-GAPDH antibodies (Santa Cruz Biotechnology, CA) were used for analyzing protein expression in SW1990 cells exogenously transfected with a plasmid encoding flag-tagged PSMC2. Blots were visualized with Electro Chemical Luminescence system, and densitometric analysis was measured with image analysis system (Thermo Fisher Scientific, MA). Values for the PSMC2 bands were normalized relative to the GAPDH bands.

Mouse anti-β-actin, mouse anti-Flag, and mouse anti-HA antibody and the following chemicals and solvents (MG132, cycloheximide, dimethyl sulfoxide (DMSO), glycerol, glycine, sodium chloride, Trizma base, and Tween20) were from Sigma (St. Louis, MO, USA). AZD1480 was from Selleckchem (Houston, TX, USA). Rabbit anti-IL4Rα, rabbit anti-IL13Rα1, mouse anti-PARP1, rabbit anti-FOXO3, mouse anti-Lamin B1, and mouse anti-GAPDH antibodies were from Santa Cruz Biotechnology. Rabbit anti-JAK2, rabbit anti-pJAK2, rabbit anti-Tyr, rabbit anti-cleaved PARP1, rabbit anti-cleaved Caspase3, rabbit anti-Bax, rabbit anti-Bim, rabbit anti-Bcl2, rabbit anti-p21, and rabbit anti-p27 antibodies were from Cell Signaling (Danvers, MA, USA). Goat anti-rabbit (111-035-003) and goat anti-mouse (115-035-003) horseradish peroxidase-conjugated IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from Genedepot (Barker, TX, USA). pECE empty/Flag-FOXO3 and pCMV3-C-HA empty/HA-JAK2 plasmid DNA were from Addgene (Watertown, MA, USA) and Sino Biological (Wayne, PA, USA), respectively.

We used a 15% SDS-PAGE gel to isolate proteins, and the proteins were transferred to a PVDF membrane. The primary antibodies used in our study were mouse anti-GAPDH (1:5000; Santa Cruz, USA), rabbit anti-RPL35 (1:100; ABclonal, China), rabbit anti-DDX10 (1:300; Proteintech, USA), mouse anti-FLAG (1:2,000; Sigma, USA), rabbit anti-RPL18 (1:100; ABclonal, China), rabbit anti-HNRNPU (1:100; ABclonal, China), rabbit anti-NCL (1:100; ABclonal, China), rabbit anti-PRMT5 (1:100; ABclonal, China), rabbit anti-E2F1 (1:100; ABclonal, China), rabbit anti-E2F4 (1:100; ABclonal, China), rabbit anti-E2F8 (1:100; ABclonal, China), goat anti-rabbit IgG (1:2,000; CST, USA), and goat anti-mouse IgG (1:2,000; CST, USA).

The cells were fixed in 4% formaldehyde in PBS for 15 min. The slides were then blocked by 10% horse serum with 0.2% Triton X-100, and co-incubated with primary antibodies (the rabbit anti-AR (Santa Cruz, sc-816), the mouse anti-GAPDH (Santa Cruz, sc-47724)) at 1:100 dilution followed by washing and incubation with secondary antibody and DAPI (Thermo Fisher) for visualization with Nikon Ti-S microscope.

Protein from spinal cord tissues and PC12 cells was extracted in an NP‐40 lysis buffer. An equal amount of protein was fractionated by 11.5% SDS‐PAGE, and transferred onto PVDF membranes (Bio‐Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% freshly prepared milk‐TBST for 90 min. at room temperature and then incubated overnight at 4°C with primary antibodies (rabbit anti‐cleaved‐caspase 3 (1:1000), goat anti‐Iba‐1 (1:1000), rabbit anti‐TNF‐α (1:1000), mouse anti‐TLR4 (1:10,000), goat anti‐IL‐6 (1:300) or mouse anti‐GAPDH (1:1000) from Santa Cruz Biotechnology; mouse anti‐NF‐κB (1:400), rabbit anti‐IκB (1:400), mouse anti‐p‐IκB (1:400) from Abcam). After being washed in TBST, membranes were incubated with appropriate secondary antibodies for 1 hr at room temperature. Proteins were detected using an enhanced chemiluminescence (ECL) kit (Bio‐Rad). Signal intensities were quantified by densitometry using Image Lab 3.0 software (Bio‐Rad). Data were normalized to total or loading controls 23, 24.