α actin

The following plasmids were used: human DVL2-GFP and FLAG-Axin (Fiedler et al., 2011 (link)); FLAG-DVL2, E499G, K446M, and D460K (Mund et al., 2015 (link)); and SNAP-FZD5 (Koo et al., 2012 (link)). DEP-RFP (red fluorescent protein) was generated by subcloning DEP into DsRed. DEP and FZD5 mutants were generated by standard procedures and verified by sequencing. The following antibodies and resins were used: α-FLAG, α-tubulin, and α-GFP (Sigma); α-actin (Abcam); and α-SNAP (NE Biolabs).

Primary antibodies against proteins of interest used in Western blot assays were: α-Tax (Tax antibodies 169, 170, and 171 monoclonal mouse (generous gift of Dr. Scott Gitlin, University of Michigan) [35 (link), 54 (link)]; α-p19 (Santa Cruz Biotechnology, sc-1665); α-gp61/46 (NIH AIDS Reagent Program Cat. # 1578); LC3-1/LC3-II (Santa Cruz Biotechnology, Cat. # SC-398822); p62 autophagy marker (Cell Signaling Inc., Cat. # 5114S); α-CD45 (Santa Cruz Biotechnology, Cat. # SC-1123); α-CD43 (Santa Cruz Biotechnology, Cat. # SC-70682); α-ICAM-1 (Santa Cruz Biotechnology, Cat. # SC-8439); α-LFA-1 (Santa Cruz Biotechnology, Cat. # SC-374172); α-CD63 (Santa Cruz Biotechnology, Cat. # SC-365604); α-CD63 tetraspanin marker (SBI System Biosciences, Cat. # EXOAB-CD63A-1); α-histone H2B (Cell Signaling Technology, Cat. # 12,364); α-histone H2A (Santa Cruz Biotechnology, Inc., Cat. # SC-10807 and abcam, Cat. # ab18255); α-histone H3 (Santa Cruz Biotechnology, Inc., Cat. # SC-10809); α-histone H4 (Cell Signaling Technology, Cat. # 2592); α-histone H1 (GeneTex, Cat. # GTX114462); IL-8 (Santa Cruz Biotechnology, Cat. # SC-376750); IL-6 (Santa Cruz Biotechnology, Cat. # SC-28343); RANTES (Santa Cruz Biotechnology, Cat. # SC-514019); GAPDH (Santa Cruz Biotechnology, Cat. # SC-48166); and α-Actin (Abcam; ab469900).

Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 15 min. The cells were then incubated with 5% normal serum that matched the species used to generate the secondary antibody and then probed with α-actin (Abcam, 1:800) primary antibody for overnight at 4°C. The cells were then incubated in fluorochrome-conjugated secondary antibodies for 2 h at room temperature. Cell nucleus was stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Merck KGaA). Images were captured with a Carl Zeiss Axioskop microscope (Carl Zeiss AG, Oberkochen, Germany). The cardiomyocyte diameter was calculated by ImageJ software [19 ].

Western blotting was carried out as described previously [17] (link). The following primary antibodies were used: for hCLE, a rabbit polyclonal antibody (1∶1000) from Abcam; for RNAP II, a monoclonal antibody 8WG16 (1∶500) from Covance; for β-tubulin, a monoclonal antibody (1∶15000) from SIGMA, for DDX-1, a goat polyclonal antibody sc-49817 (1∶200) from Santa Cruz Biotechnology; for HSPC117/C22orf28, a goat polyclonal antibody LS-C139785 (1/3000) from LifeSpan BioSciences; for FAM98B, a rabbit polyclonal antibody HPA008320 (1/500) from Sigma; for α-actin, a rabbit polyclonal antibody (1/200) from Abcam and for S6, a mouse monoclonal antibody 54D2 (1/1000) from Cell Signaling that recognizes total S6 protein.

Rat ADM (molecular formula: C₂₄₂H₃₈₁N₇₇O₇₅S₅) was obtained from Bachem (Bubendorff, Switzerland). PA and Compound C were purchased from Sigma Aldrich (St. Louis, MO, USA). ADM22-52 was purchased from Anaspec (Fremont, CA, USA). DMEM, 0.25% trypsin-EDTA, fetal bovine serum, trypsin and streptomycin/penicillin were from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibodies against α-actin and Runx2 were purchased from Abcam (Burlingame, CA, USA). ADM, CRLR, RAMP2, RAMP3, TNF-α and IL6 were obtained from Affinity Biosciences (Pottstown, PA, USA). The antibodies of IL-1β, NOX2, NOX4 and GAPDH were from Proteintech (SANYING, Wuhan, China).

Whole cell extracts for Western blotting were prepared as described previously (16 (link)). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon-FL Polyvinylidene fluoride (PVDF) membranes (Millipore) according to standard procedures (Novex). Blots were probed with following antibodies: XRCC1 (Neomarkers, MS-1393-P0), DNA polymerase β (raised in-house and affinity-purified), α-tubulin (Sigma, T6199), MCM4 (abcam, ab4459-50), RP-A p32 (Bethyl, A300-244A), PCNA (Santa Cruz, sc-56), α-actin (Abcam, ab6276), PARP-1 (raised in-house and affinity-purified), p21 (Cell signaling, 12D1), PSAT1 (Novusbio, 21020002), PHGDH (Sigma, HPA021241), p53 (Santa Cruz, sc-126), PNKP (Abnova, H00011284-B01). Secondary antibodies conjugated with Alexa Fluor 680 (Molecular Probes) and IRDye^® 800 (Rockland) fluorescent dyes were used. Detection and quantification was carried out using an Odyssey image analysis system (Li-Cor Biosciences).