Gapdh

Antibodies against chlamydia (C. trachomatis, C. psittaci, and C. abortus), chlHSP60 (MAb A57-B9), cellular β-tubulin, and GAPDH, were obtained from Acris, Sigma-Aldrich, and Abcam. In addition, the fluorescein isothiocyanate (FITC)-conjugated mouse anti-chlamydia MAb (part of the IMAGEN Chlamydia Kit) was purchased from Oxoid. All of the secondary and isotype-control antibodies were purchased from Dianova and BioLegend.

The total RNA was extracted using TRIzol reagent (Invitrogen Inc., Carlsbad, CA, United States). Reverse transcription of miRNA was performed using a transcription kit (Shanghai GeneChem Co., Ltd., Shanghai, China) with RNA U6 as an internal control. mRNA was detected using a reverse transcription kit (Takara Biotechnology Ltd., Dalian, Liaoning, China) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence as an internal reference for the normalization of RT-PCR. SYBR Green quantitative PCR analysis was performed using a 7500 real-time fluorescence quantitative PCR system.
The expression levels of target genes were then estimated using the 2^−ΔΔCT method. ^{17 (link)
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Primers STAT1 (#HP210040), JAK2 (#HP208201), IRF-1 (HP205934), PD-L1 (#HP210654), IFN-γ (#HP200586), NF-κB (#HP207409), Bcl-xL (#HP234144), COX-2 (#HP200900), GAPDH (#HP205798), and β-catenin (#KN208947) were purchased from OriGene Technologies (Beijing, China).
The sequence of primers is presented in table 1.

Proteins were extracted from renal tissues or cultured cells with a RIPA lysis buffer and analyzed via Western blotting. Briefly, after the protein was transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA), the membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS/T) for 1 h. The membranes were incubated with primary antibodies against phosphor-p53 (Ser 15) and P53 (Novus Biologicals), cleaved-caspase-3 (Cell Signaling Technology) and GAPDH (Origene) overnight at 4 °C. The membranes were washed three times with 10 mL of TBS/T and then incubated with secondary antibodies (R&D Systems) for 1 h at room temperature. After the membranes were washed three times with TBS/T, the proteins on the membrane were visualized with an enhanced chemiluminescence reagent (Millipore Corporation, Boston, MA, USA). The signals were detected with an Odyssey Infrared Imaging System (Bio-Rad, ChemiDoc MP, mANUSC, Bio-Rad Laboratories Inc., Hercules, CA, USA) and quantified with the Image J program (National Institutes of Health). The ratio for the protein examined was normalized against GAPDH.

Treated HASMCs and PBMNCs were lysed in RIPA buffer and protein content was determined using a BCA Protein Assay Kit (cat. P0011, Beyotime). Proteins were separated by 12% or 9% SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). Primary antibodies against WNT3 (cat. GTX89319; Gene Tex, CA, USA), β-catenin (cat. GTX101435-S; Gene Tex), calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2, cat. TA322113S; Origene, MD, USA), and GAPDH (cat. TA-08; ZSGB-BIO, Beijing, China) were applied at 4 °C overnight. Subsequently, a horseradish peroxidase-conjugated secondary antibody (1:10000) was applied at room temperature for 1 h. The signals were detected with a SuperSignal West Femto Trial Kit (Thermo Fisher Scientific). Relative protein content was analyzed by ImageJ software and normalized to loading controls.