Ab108596

Tissues or cardiomyocytes were sonicated in RIPA lysis buffer containing 1% NonidetP-40, 0.1% SDS, protease inhibitor, while adding phosphatase inhibitors cocktail (Bimake, Houston, USA), and then homogenized. The nuclear and cytosolic protein were extracted by commercially available kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells expanded sufficiently under low osmotic pressure, then the cell membrane ruptured, releasing cytoplasmic proteins. Nucleus precipitation could be centrifuged to obtain after then. Finally, the nuclear protein was extracted by high salt nucleoprotein extraction reagent. Equal quantities of tissues or cardiomyocytes protein lysates were subjected to SDS-PAGE (Bio-Rad), and transferred onto PVDF membrane. Blocking was made at room temperature with 5% nonfat milk powder prepared in Tris-buffered saline containing 0.1% Tween 20. Then, membranes were incubated overnight at 4 °C with corresponding antibodies, and followed by incubation with appropriate secondary HRP-conjugated antibodies. Antibodies against p38 (#8690), P-p38 (#4511), NFκB-p65 (#8242), ERK (#4695), P-ERK (#4370), P-Stat3 (#9145), Stat3 (#4904) and GAPDH (#2118) were obtained from Cell signaling Technology (Danvers, USA). Antibodies against FNDC5 (ab174833), P-JAK2 (ab195055), JAK2 (ab108596), Nox2 (ab129068), Nox4 (ab154244) were obtained from Abcam (Cambridge, UK).

Total proteins were extracted after cell lysis with the radioimmunoprecipitation assay lysis buffer (RIPA; Accuref Scientific, Xi’an, China). A total of 20 μg samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with tris-buffered saline (TBS) supplemented with 5% nonfat dry milk for 1 hour, the membrane was incubated with the indicated primary antibodies (anti-PRLR, 1:1000 dilution, #ab170935; anti-JAK2, 1:5000 dilution, #ab108596; anti-pSTAT3, 1:1000 dilution, #AB267373; anti-β-actin, 1 μg/ml, #ab8226; all purchased from Abcam) at 4°C overnight. After binding with the secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:10,000; #ab6721, Abcam) or anti-mouse IgG (1:10,000; #ab6728, Abcam) for 1 hour at room temperature, the membranes were processed with the enhanced chemiluminescence kit (Accuref Scientific, Xi’an, China). The expressions of targeted proteins were quantified by densitometry using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The cytoplasm and nuclear fraction of cells were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions. Whole cell lysates or the nuclear/cytoplasm fractions were subjected to SDS-PAGE and immunoblotting, as described in our previously published study [24 (link), 25 (link)]. Primary antibodies against STAT3 (Abcam, ab119352), phosphorylated STAT3 (p-STAT3) (Abcam, ab76315), GAPDH (Abcam, ab181602), GNAS (Proteintech, 10,150–2-AP), YTHDF1 (Abcam, ab220162), YTHDF2 (Abcam, ab220163), YTHDF3 (Abcam, ab220161), P65 (Proteintech, 10,745–1-AP), phosphorylated P65 (pp65) (Abcam, ab76302), JAK1 (Abcam, ab133666), and JAK2 (Abcam, ab108596) were used.

HK-2 cells and kidney tissues were homogenized in radioimmunoprecipitation assay buffer (Beyotime, Nanjing, China) containing protease inhibitors to lyse the cells to obtain total proteins. After collecting the proteins in each sample separately, the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes in an ice-water bath and incubated with primary antibody at 4 °C for 12 h after being closed with 5% skim milk for 1 h at room temperature. Primary antibody was used at the following dilutions: Bax (1:200, Cell Signaling, 2772); Bcl-2 (1:1000, Abcam, Ab196495); CHOP (1:1000, Cell Signaling, 2895); GRP78 (1: 1000, Abcam, Ab21685) and GAPDH (1:1000, Hangzhou Jiahe Biotechnology Co., AB-PR 001);JAK2(1:5000, Abcam, Ab108596);p-JAK2(1:1000, Abcam, Ab32101);STAT3(1:1000, Abcam, Ab68153);p-STAT3(1:1000, Abcam, Ab267373). The membranes were then washed three times with TBST for 20 min each, incubated for 2 h at room temperature with appropriate secondary antibodies, then washed three times with TBST for 10 min each, and finally the western blots were visualized using chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). Data analysis was performed using Image J software (NIH, USA) to quantify protein levels.