Mouse monoclonal anti v5 antibody

To confirm the location of APP770-V5 and FNDC5-HA, we examined immunofluorescence staining of SH-SY5Y cells transiently expressing these molecules. We washed prepared cells by PBS and fixed them by 4% paraformaldehyde for 15 min at room temperature. Then, these cells were permeabilized by 0.1% Triron-X and blocked by using Blocking solution (Nacalai Tesque, Japan). We used the mouse monoclonal anti-V5 antibody (1:1000; Sigma) and the rabbit polyclonal anti-HA antibody (1:1000; Sigma) for the primary antibodies to detect APP and FNDC5, and then labeled them by Alexa Fluor 594-conjugated goat anti-mouse (1:2000; Life Technologies, MA, USA) and Alexa Fluor 488-conjugated mouse anti-rabbit (1:2000; Life Technologies), respectively. As the mounting agent, we used NucBlue Fixed Cell Stain ReadyProbes reagent from Life Technologies. These cells were observed using a laser confocal scanning microscope (FV10i-LIV, Olympus, Japan).

The following primary antibodies were used: (1) rabbit polyclonal antibodies to PITX2A,B,C (Capra Science, Ängelholm, Sweden; at 1∶2000 dilution), (2) rabbit polyclonal antibodies to PITX2B (Capra Science, Ängelholm, Sweden; at 1∶1000 dilution).The specificity of the anti-PITX2 antibodies was independently validated in this study by Western blot analysis of COS-7 cells expressing each PITX2 isoform (see Fig 1 A, B), (3) rabbit monoclonal antibodies to MYF5 (Abcam, Cambridge, UK; at 1∶10000 dilution), (4) rabbit polyclonal antibodies to cardiac troponin I (Abcam, Cambridge, UK; at 1∶40000 dilution), (5) rabbit polyclonal antibodies to cardiac calsequestrin-2 (Abcam, Cambridge, UK; at 1∶10000 dilution), (6) rabbit polyclonal antibodies to ANKRD1 (at 1∶1000 dilution) were generated by Davids Biotechnologie (Regenburg, Germany) using the N-terminal region of pig ANKRD1 as immunogen [22] (link), (7) mouse monoclonal anti-FLAG M2 antibody (Sigma, Madrid, Spain; at 1∶5000 dilution), (8) mouse monoclonal anti-V5 antibody (Sigma, Madrid, Spain, at 1∶5000 dilution), and (9) mouse monoclonal anti-GAPDH antibody (Sigma, Madrid, Spain, at 1∶10000 dilution). Secondary peroxidase conjugated anti-rabbit and anti-mouse IgG (Fab-specific) antibodies were purchased from Sigma (Madrid, Spain).

S. cerevisiae strains (Supplementary Table 1) were derived from BY4741 (ref. 62 (link)) using standard methods. The dna2-2 allele was generated using pop-in/pop-out mutagenesis63 (link), and the DNA damage sensitivity of the resulting strain could be complemented with plasmid-borne wild-type DNA2 cloned into vector pAG416GPD-ccdB (pDNA2) (Supplementary Fig. 2b). For constitutive expression, YEN1 was cloned into vector pAG416GPD-ccdB or pAG416GPD-ccdB-EGFP64 (link), and site-directed mutagenesis was performed to generate a catalytically inactive form of Yen1 bearing the mutations E193A and E195A. Yellow fluorescent protein (YFP)-tagged Rad52 was expressed from its endogenous promoter using centromeric plasmid pWJ1213 (ref. 65 (link)). If not stated otherwise, all strains were cultured at 30 °C using YPAD media. YEN1^on was cloned into vector pAG416GPD-ccdB, or pYES-DEST52 (Invitrogen) for expression from a GAL1 promoter in YPLG medium with 2% (w/v) galactose and 1% (w/v) raffinose. Antibodies used to monitor the expression of tagged proteins were Abcam mouse monoclonal anti-V5 antibody ab27671, and Sigma mouse monoclonal anti-Myc antibody 9E10. Santa Cruz Biotechnology goat polyclonal anti-Mcm2 antibody yN-19 was routinely used to ensure gel lanes were equally loaded for total protein.