Anti jnk

Samples and pre-stained standard separated on a 4–12% NuPAGE™ (Invitrogen) were transferred onto a piece of Immobilon™ membrane (Millipore, Bedford, MA). The membranes were rinsed with TBS-T (TBS containing 0.05% Tween 20), blocked with 5% nonfat dry milk at room temperature for 1 h, and incubated with appropriately diluted (1:500 ~ 2000) 1st antibodies overnight at 4 °C. The first antibodies were rabbit IgG purchased from Cell Signaling (Beverly, MA), including anti-I-κBα (#9242), anti-GAPDH (#2118), anti-phospho-p44/42 (#9101, Thr202/Tyr204), anti-p44/42 (#9102), anti-phosphorylated p38 (#9211, Thr180/Tyr182), anti-p38 (#9212), anti-phosphorylated JNK (#9251, Thr183/Tyr185), anti-JNK (#9252), anti-phosphorylated p53 (#9284, Ser15), anti-Cdc2 (#77,055), and anti-cyclin B1 (#4138). After washing three times with TBS-T, the membranes were reacted with 1:2000 diluted HRP-conjugated goat anti-rabbit IgG (Cell Signaling, #70,741) for 1 h at room temperature, washed, and developed in SuperSignal^® West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc., Hanover Park, IL). The images were collected using the G BOX Chemi systems (Syngene, Frederick, MD).

Total proteins of BMMSCs were isolated using RIPA (Cell Signaling Technology, USA). The automated western blot was performed using Simple Wes (Protein Simple, USA) following the manufacturer’s protocol. Briefly, 2.5 μg of protein from the cell lysates was added to the standard fluorescent mastermix, then was loaded into corresponding wells of the prefilled Wes assay plate, along with antibody diluent (Protein Simple, USA), anti-MagT1 (Proteintech, USA), anti-DSP (Santa Cruz, USA), anti-DMP-1 (Genetex, USA), anti-ALP (Affinity, USA), anti-ERK (Cell Signaling Technology, USA), anti-p-ERK (Cell Signaling Technology, USA), anti-JNK (Cell Signaling Technology, USA), anti-p-JNK (Cell Signaling Technology, USA), anti-p38 (Cell Signaling Technology, USA), anti-p-p38(Cell Signaling Technology, USA), anti-β-Tublin (Affinity, USA), anti-rabbit secondary antibody (Protein Simple, USA), and Streptavidin-HRP, followed by luminal peroxide mix. The imaging and analysis were done with compass software (Protein Simple).

Immunoblotting was carried out as described previously [11 (link)]. The following antibodies were purchased from Abcam: anti-GAPDH (1:2500, 37 kDa, AB9485, RRID:AB_307275), anti-IL-6 (1:2000, 17 and 50 kDa, ab9324, RRID:AB_307175), and anti-ICAM-1 (1:4000, 90 kDa, AB53013, RRID:AB_870702). Anti-ACE2 (1:1000, 125 kDa, MA532307, RRID:AB_2809589) and anti-HIF-1α (1:1000, 130 kDa, PA5-85,494, RRID:AB_2792634) antibodies were obtained from Thermo Scientific. The following antibodies were purchased from Cell Signaling Technology: anti-ERK1/2 (1:1000, 42, 44 kDa, #4695, RRID:AB_390779), anti-phospho-ERK1/2 (Thr202/Tyr204), (1:1000, 42, 44 kDa, # 9101, RRID:AB_331646), anti-MSK1 (1:1000, 95 kDa, #3489, RRID:AB_2285349), anti-phospho-MSK1 (Thr581) (1:1000, 95 kDa, #9595, RRID:AB_2181783), anti-p38 (1:1000, 43 kDa, #9212, RRID:AB_330713), anti-phospho-p38 (Thr180/Tyr182) (1:1000, 43 kDa, #4511, RRID:AB_2139682), anti-phospho-JNK (Thr183/Tyr185) (1:1000, 46, 54 kDa, #4668, RRID:AB_823588), anti-JNK (1:1000, 46, 64 kDa, #9252, RRID:AB_2250373), anti-phospho-NFκB p65 (Ser536) (1:1000, 70 kDa, #3033, RRID:AB_331284), and anti-NFκB p65. (1:1000, 70 kDa, #6956, RRID:AB_10828935). The following antibody were obtained from ABclonal (Woburn, MA): anti-phospho-NFkB p65 (S276) (1:1000, 70 kDa, AP0123, RRID:AB_2771505). An anti-phospho-MBP antibody (1:200, 18 kDa, 13–104) was purchased from Merck.

In this study, the antibodies respectively against p38, p-p38, ERK, p-ERK, Akt1/2/3, p-Akt1/2/3, OGT, OGA, CD68 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal Anti-β-O-Linked N-Acetylglucosamine Clone CTD110.6 was obtained from Sigma (St. Louis, MO, USA). Other antibodies including anti-JNK, anti-p-JNK, Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).
Chemicals such as 1-phenyl-3-methyl-5-pyrazolone (PMP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris, Tween 20, trifluoroacetic acid, LPS (from Escherichia coli 055:B5), 6-diazo-5-oxo-L-norleucine (DON) and bovine serum albumin were obtained from Sigma. The ABC kit and Agarose wheat germ agglutinin (WGA) were purchased from Vector Laboratories (Lowellville, OH, USA). Thiamet G (Thi G) was obtained from Selleck Chemicals (Houston, TX, USA). RPMI 1640 was purchased from Corning Incorporated (Corning, NY, USA), penicillin, streptomycin and heat-inactivated fetal bovine serum (FBS) was from Gibco (Grand Island, NY, USA). All the other chemicals including sodium dodecyl sulfonate, ammonium persulfate, isopropanol, hydrochloric acid, glycine, sodium chloride and ammonia were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

The cells were treated with TNFα (10 ng/ml) and lysed in lysis buffer (1X PBS, pH 7.4, 10 mM dithiothreitol, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail) containing 2 mM Na₃VO₄, 10 mM Na₄P₂O₇, and 10 mM NaF. Equal amounts of lysate were prepared and immunoblotted with anti-JNK (1:1000; Cell Signaling Technology, Beverly, MA) and anti-phospho-JNK (1:1000; Cell Signaling Technology) antibodies.

Protein extraction was carried out by lysing cells in PBS buffer containing 1% Triton X-100 with added protease and phosphatase inhibitors (Roche). Protein content was determined using Bradford assay (Pierce). Approximately 25 to 70 μg of total protein was resolved on 8, 10 or 12% SDS-PAGE gel before transferring onto PVDF membrane (Millipore) for detection using ECL reagent (Pierce). The following antibodies were used: anti-phospho-JNK, anti-JNK, anti-phospho-MKK4, anti-MKK4, anti-FTH1 (Cell Signaling Technology), anti-TRX (Santa Cruz), anti-LC3B (Abgent), anti-SOD2 (Abcam), anti-actin and HRP-conjugated anti-rabbit and anti-mouse (Sigma).

Letrozole (4,4-(1H-1,2,4-Triazol-1-ylmethylene) bisbenzonitrile) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Recombinant RANKL and M-CSF were obtained from PeproTech (Rocky Hill, NJ, USA), and the antibodies against NFATc1 and c-Fos were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The other antibodies (anti-phosphor-IKK, anti-IKK, anti-phosphor-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phosphor-p38, and anti-p38) were purchased from Cell Signaling Technology (Danvers, MA, USA). The leukocyte acid phosphatase kit (TRAP staining kit) and antibody for β-actin were obtained from Sigma-Aldrich. All other reagents were purchased from Sigma-Aldrich.