Ab2000

The cells and liver tissues were lysed with RIPA containing completed protease and phosphatase inhibitor, collected for centrifugation. The protein concentration was measured by a BCA Protein Assay Kit (Pierce Biotechnology, Waltham, MA). After denaturation with loading buffer, the cell lysates were separated with 10%-15% sodium dodecyl sulfate (SDS) –polyacrylamide gel electrophoresis (PAGE) gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% (m/v) skim milk and incubated overnight at 4°C with primary antibodies: CX3CR1(1 : 1000, Proteintech,13885-1-AP), CX3CL1 (1 : 1000, Proteintech, 10108-2-AP), and GAPDH (1 : 10000, Abways, AB2000). Then, the membrane was washed 3 times in TBST (10 min. each time) at room temperature and incubated with secondary antibodies: goat-anti-mouse (1 : 10000, Abways, AB0102) and goat-anti-rabbit (1 : 10000, Abways, AB0101) at room temperature for 1 hour. Finally, the membrane exposure was performed using enhanced chemiluminescence (ECL) by the Bio-Rad system.

Protein from heart tissues and cardiomyocytes were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to poly vinylidene fluoride (PVDF) membranes (Roche, Switzerland). The blotted membranes were incubated with antibodies against cleaved-caspase-3 (#9664, Cell Signaling Technology, United States), p53 (#2524, Cell Signaling Technology), phospho-p53 (#12571, Cell Signaling Technology), Bax (ab182733, Abcam, United States), bcl-2 (ab59348, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AB2000, Abways, China). Immunoreactivity was detected using an enhanced chemiluminescence reaction system (Universal Hood II, Bio-Rad, United States). Each experiment was performed in triplicate.

MSC-exos, cells and kidney tissues were lysed in RIPA lysis buffer (ThermoFisher) and the protein concentration was detected using BCA assay (ThermoFisher). Protein samples were subjected to Bis-Tris Gel (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 10 min and incubated overnight with primary antibodies as follows: anti-CD9 (ab92726, Abcam), anti-CD63 (sc-5275, Santa Cruz), anti-Tsg101 (sc-7964, Santa Cruz) anti-Alix (sc-53540, Santa Cruz), anti-GM130 (12480, Cell Signaling Technology), anti-CD44 (ab189524, Abcam), anti-ICAM-1 (MA5407, Invitrogen), anti-VCAM-1 (39036, Cell Signaling Technology), anti-LFA-1 (ab13219, Abcam), anti-integrin α4 (8440, Cell Signaling Technology), anti-integrin β1 (sc-374429, Santa Cruz), anti-PCNA (sc-56, Santa Cruz), anti-CDK1 (sc-54, Santa Cruz), anti-caspase-3 (ab13847, Abcam), anti-Cyclin B1(4138, Cell Signaling Technology), anti-p53 (2524, Cell Signaling Technology), anti-Bcl-2 (sc-7382, Santa Cruz), and anti-Bax (sc-20067, Santa Cruz). Secondary antibodies were used for detection by an ECL advanced system (GE Healthcare). Intensity values expressed as the relative protein expression were normalized to β-actin (AB2001, Abways) or GAPDH (AB2000, Abways).

Antibodies for ATM (2873S; Cell Signaling; 1:1000), 53BP1 (NB100-304; NOVUSBIO; 1:1000), RIF1 (ab1213422; Abcam; 1:500), REV7 (A9861; Abclonal; 1:1000), REV1 (sc-393022; Santa Cruz; 1:1000), REV3L (GTX17515; Gene Tex; 1:1000), AID (A16217; Abclonal; 1:1000), MSH2 (ab227941; Abcam; 1:1000), β-actin (AC028; Abclonal; 1:10,000), FLAG (F1804, Sigma; 1:1000), β-Tubulin (A01030HRP; Abbkine; 1:10,000), glyceraldehyde 3-phosphate dehydrogenase (AB2000; Abways; 1:20,000), and Rabbit TrueBlot (18-8816-33; Ebioscience; 1:1000) were used in western blotting. PE-conjugated anti-mouse IgA (12-4204-83; Ebioscience; 1:200), APC-conjugated anti-mouse IgM (1020-11S; Southern biotech; 1:200), APC-conjugated anti-mouse B220 (553092, BD; 1:200), fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG1 (553443; BD; 1:200), FITC-conjugated anti-mouse IgG3 (553403; BD; 1:200), APC-eFluor780-conjugated anti-mouse B220 (47-0452-82, Invitrogen; 1:200), FITC-conjugated anti-mouse GL7 (144604, BioLegend; 1 : 200), PE-Cy7-conjugated anti-mouse CD95 (557653, BD; 1:200), and Fluorescein-labeled Peanut Agglutinin (FL-1071, Vector Laboratories; 1:500) were used in the fluorescence-activated cell sorting analysis.

The protein lysates from exosomes or kidney tissues were prepared following standard protocols, and the protein content was determined using the BCA protein assay kit. Then, protein samples were separated by Bis-Tris Gel (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% BSA in TBST for 1 h at room temperature and were incubated with primary antibodies overnight at 4 °C. Then, membranes were washed three times and incubated with secondary antibodies for 2 h at room temperature, and the signals were detected using an ECL advanced system (GE Healthcare). Intensity values expressed as the relative protein expression were normalized to GAPDH (AB2000, Abways). Primary antibodies used were anti-Alix [35 (link)] (sc-53540, Santa), anti-CD63 (ab193349, Abcam), anti-CD9 (ab92726, Abcam), and anti-phosphorylated NF-κB p65 (3033, Cell Signaling Technology). Secondary HRP-conjugated antibodies used were anti-mouse IgG and anti-rabbit IgG (Abcam).

Total RNA and protein were isolated with TRIzol reagent according to manufacturer's instructions (Invitrogen). Then, polyA⁺ RNA was reversely transcribed into cDNA with oligo‐(dT) primer (AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN) using FastQuant RT Kit (Tiangen). Gene expression at the RNA level was quantified by qRT‐PCR using 2× SYBR mix (Vazyme). GAPDH served as an internal control. Then, the reaction was run on Bio‐Rad CFX manager machine.
For Western blot, protein was incubated at 95°C for 10 min before performing SDS‐PAGE. The primary antibodies for U2AF1 (ab86305; 1: 2000, Abcam), CPNE1 (ab155675, 1: 1000, Abcam), and GAPDH (AB2000, 1: 5000, Abways) were incubated with membrane for 2 h at room temperature. The blots were then washed and incubated with second antibody (Goat Anti‐Rabbit, M21002L, 1: 5000, Abmart) for 1 h at room temperature. After washing in TBST, blots were exposed by Tanon Chemiluminescent Imaging System.

After treatment with JDB175 for 48 h, the cells were harvested and protein lysates were prepared in RIPA buffer containing proteinase inhibitor cocktail. Subsequent western blot analyses were performed using the indicated primary antibodies and secondary antibodies conjugated to horseradish peroxidase. The protein bands were developed after incubating with chemiluminescent substrate (Amersham Biosciences). Quantification of the band intensities was based on three independent experiments using ImageJ software. Antibodies for western blot analysis were purchased from Cell Signaling Technology (anti‐AKT, 4685S, 1:1000; anti‐p‐γ‐H2AX, 9718S, 1:1000; anti‐cleaved caspase3, 9664S, 1:1000), ABclonal Technology (anti‐γ‐H2AX, A11463, 1:1000), and Abways (BTK, CY5733, 1:1000; p‐BTK, CY5558, 1:1000; p‐p44/42 MAPK, CY5044, 1:1000, p44/42 MAPK, AB3373, 1:1000; p‐AKT, CY6569, 1:1000; GAPDH, AB2000, 1:10,000; BAX, CY5059, 1:1000; cyclin D1, CY5404, 1:1000; CDK2, CY5020, 1:1000; p21, CY5543, 1:1000).