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2 protocols using αha 16b12

1

Comprehensive Antibody Characterization for Molecular Biology

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Antibodies used in this study include αm6A (Millipore, ABE572 or MABE1006) for MeRIP/m6A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH. Other antibodies that were applied in Western blot analysis include αMETTL3 (Proteintech, 15073-1-AP), αMETTL14 (Sigma-Aldrich, HPA038002), αALKBH5 (Sigma-Aldrich, HPA007196), αHA (16B12) (BioLegend, #901501), αmyc (9E10) (Santa Cruz Biotechnology, sc-40), αHis (Proteintech, #66005-1), αCRISPR-Cas9 (Abcam, ab191468), αAR (H-280) (Santa Cruz Biotechnology, sc-13062), αYTHDF2 (Proteintech, 24744-1-AP), αYTHDC2 (Proteintech, 27779-1-AP), αp53 (DO-1) (Santa Cruz Biotechnology, sc-126), αp21 (Cell Signaling Technology, #2947), αMDM2 (Proteintech, 19058-1-AP), αTelomerase reverse transcriptase (TERT) (Abcam, ab32020), and αβ-actin (Sigma-Aldrich, A5441). Reagents that were used in IP, ChIP, and MeRIP are Protein A/G Plus Agarose (Santa Cruz Biotechnology, sc-2003) and Dynabeads Protein A (Thermo Fisher Scientific, #10006D) or Protein G (Thermo Fisher Scientific, #10007D) immunoprecipitation kit.
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2

Immunoblot Analysis of Yeast Proteins

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Proteins were separated by SDS-PAGE (10% gels) followed by immunoblot analysis using mouse monoclonal antibodies α-Myc (9E10; Santa Cruz), α-HA (16B12; BioLegend), α-Carboxypeptidase Y (CPY; 10A5B5; Abcam), or goat polyclonal α-Mpk1 (yC-20; Santa Cruz) at a dilution of 1:10,000. Rabbit polyclonal α-phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling), was used at a dilution of 1:2,000 to detect phosphorylated Mpk1. Goat polyclonal α-Hog1 (yC-20; Santa Cruz) at a dilution of 1:10,000, or rabbit polyclonal α-phospho-p38 (T180/Y182, Cell Signaling) at a dilution of 1:2,000 were used to detect Hog1 and phosphorylated Hog1, respectively. Secondary goat anti-mouse (Jackson ImmunoResearch Labs), donkey anti-rabbit (GE Healthcare), or mouse anti-goat (Santa Cruz) antibodies were used at a dilution of 1:10,000. All results involving immunoblot analyses were replicated at least once and representative blots are shown.
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