Anti p jak2

Proteins from AR42J cells were extracted using radioimmunoprecipitation assay lysing solution and quantified using the Bicinchoninic acid (BCA) methods. Separated from 12% gel with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. After inhibition with 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. On the next day, the secondary antibody was applied to incubate the membranes for 1 h. Finally, the protein bands were imaged by enhanced chemiluminescence (Invitrogen; Thermo Fisher Scientific, Inc.). The following primary antibodies were used: anti-FXYD5 (1:500; Proteintech^TM), anti-Bcl-2 (1:1000; Abcam), anti-Bax (1:1000; Abcam), anti-Cox2 (1:1000; Abcam), anti-iNOS (1:1000; Abcam), anti-p-JAK2 (1:1000; Abcam), anti-JAK2 (1:2000; Abcam), anti-p-STAT3 (1:1000; Abcam), anti-STAT3 (1:1000; Abcam) and anti-GAPDH (1:500; Abcam).

Forty-eight BALB/c mice (half male and half female; body mass 22–28 g) were purchased from the Experimental Animal Center of Lanzhou University (No. SCXK (Gan) 2018-0002). The mice were housed at a temperature of (25 ± 1)°C, relative humidity of 65% ± 10%, and a 12 hr light–dark cycle. All studies were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and in compliance with the regulations of the Gansu Provincial Animal Management Committee. All necessary efforts were made to minimize animal suffering and reduce the number of animals used in the experiments.
Lipopolysaccharide and morroniside were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Anti-iNOS, anti-COX2, anti-Arg-1, anti-CD206, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3, and goat anti-rabbit IgG-HRP antibodies were purchased from Abcam (Cambridge, UK). TNF-α, IL-6, and IL-1β ELISA kits were purchased from Shanghai ZCIBIO Technology Co., Ltd. PBS and trypan-blue solution were purchased from Beijing Solarbio Technology Co., Ltd. A bicinchoninic acid assay (BCA)-100 Protein Quantitative Analysis Kit was purchased from Shanghai Biocolor Biotechnology Co., Ltd. RIPA and BCA protein assay kits were purchased from Beyotime, Beijing. ECL was purchased from Affinity, Shanghai. AG490 was purchased from Shanghai Zerun Bio.

An STAT3 inhibitor (CAS no.14556632-40-8; product no. SH-4-54; purity >98%) was purchased from Med Chem Express Co., Ltd. (USA). Anti-MMP-9 (catalog no. # ab38898), Anti-P-STAT3 (catalog no. # ab32143), STAT3 (catalog no. # ab68153) Anti-P-JAK2 (catalog no. # ab32101), JAK2 (catalog no. # ab108596), and anti-β-actin (catalog no. # ab8229) antibodies were obtained from Abcam (Cambridge, MA, USA). The sequences of the forward and reverse primers for MMP-9 and GAPDH were designed using primer 3 and synthesized by Jinsirui Co., Ltd. (Nanjing, China). The IL-6 ELISA kit (catalog no. # EK0410) was purchased from Boster Biotechnology Co., Ltd. (Wuhan, China). The QIAGEN miRNeasy mini kit (catalog no. # 217004) was obtained from QIAGEN (Germany). The TaKaRa PrimeScript RT reagent kit (catalog no. #RR036A) and TaKaRa TB Green Premix Ex Taq (catalog no. #RR820A) were purchased from TaKaRa (Japan). ProLong™ Gold antifade reagent (P36931) was obtained from Invitrogen (Thermo Fisher Scientific, USA). The CMS rat model was established in an automatically adjusted low-pressure hypobaric chamber (DYC-300, Guizhou Fenglei Oxygen Chamber Co., Ltd., Guizhou, China).

The cell extracts were prepared using RIPA buffer (KeyGen Biotech) containing protease inhibitors (KeyGen Biotech). Equal amounts of protein samples were subjected to 12% SDS-PAGE and transferred to PVDF membranes (Immobilon-P; Millipore, Billerica, USA). The membranes were then blotted with primary antibodies overnight at 4°C, followed by incubation with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000). The antibodies used were as follows: anti-AIFM2 (1:1000; Biorbyt, Cambridge, UK), anti-Bcl2 (1:2000; Abcam), anti-Bax (1:5000; Abcam); anti-Caspase3 (1:5000; Abcam), anti-Bak (1:1000; CST), anti-Cytc (1:1000; Abcam), anti-p53 (1:2000; Abcam), anti-p-mTOR (1:1000; CST), mTOR (1:1000; CST), anti-p-PI3K (1:1000; Affinity Biosciences), anti-PI3K (1:1000; Affinity Biosciences), anti-p-AKT (1:1000; CST), anti-AKT (1:1000; CST), anti-p-JAK2 (1:1000; Abcam), anti-JAK2 (1:1000; Abcam), anti-p-STAT3 (1:5000; Abcam), anti-STAT3 (1:5000; Abcam) and anti-GAPDH (1:5000; BBI, Shanghai, China). The protein bands were then visualized using enhanced chemiluminescence reagent (Bio-Rad, Hercules, USA). Band quantification was conducted using ImageJ (National Institutes of Health, Bethesda, USA).

Cells were lysed using a RIPA buffer, including a protease inhibitor cocktail (Thermo Scientific, USA). The proteins were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies at 4 °C overnight. The HRP-conjugated secondary antibodies were used to incubate the membranes for 2 h at room temperature. The membranes were washed and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. Proteins were detected using a Bio-Rad ChemiDoc XRS + System. Bio-Rad Image Lab software was used for densitometric analysis. The following primary antibodies were purchased: anti-E-cadherin (1:1000; Cell Signaling, USA), anti-Vimentin (1:1000; Proteintech, USA), anti-p-JAK2 (1:1000; phosphor Y1007 + Y1008) (1:1000; Abcam, USA), anti-JAK2 (1:1000; Abcam, USA), anti-p-STAT3 (phosphor Y705) (1:1000; Cell Signaling, USA), anti-STAT3 (1:1000; Cell Signaling, USA), anti-p-AKT (phosphor S473) (1:1000; Abcam, USA), anti-AKT (1:1000; Abcam, USA), anti-p-ERK1/2 (phosphor T202 + T204) (1:1000; Cell Signaling, USA), anti-ERK1/2 (1:1000; Cell Signaling, USA), anti-FoxQ1 (1:1000; Sigma-Aldrich, USA), anti-GAPDH (1:5000; Santa Cruz, CA), anti-β-actin (Santa Cruz, CA).