Western Blot:Article Title: PRKN ‐Mediated Ubiquitin‐Proteasome Degradation of METTL3 Promotes Cellular Senescence
Article Snippet: whole‐cell lysates were incubated overnight with the appropriate antibodies, followed by incubation with Protein A/G beads (sc‐2003, SANTA CRUZ), or anti‐Flag beads (Sigma). .. The following antibodies were used for immunoblotting: anti‐METTL3 (1:2000, ab195352, Abcam), anti‐METTL14 (1:3000, 26158‐1‐AP, Proteintech), anti‐WTAP(1:5000, 10200‐1‐AP, Proteintech), anti‐PRKN (1:1000, 14060‐1AP), anti‐p16 (1:1000, 10883‐1‐AP, Proteintech), anti‐p21 (1:1000, 2947S, CST), anti‐TRF1 (1:500, T1948, Sigma‐Aldrich), anti‐TRF2 (1:1000 dilution, 66893‐1‐IG, Proteintech), anti‐POT1 (1:500 dilution, 10581‐1‐AP, Proteintech), HRP‐conjugated goat anti‐rabbit (1:2000, 7074S, Cell Signaling Technology) or horse anti‐mouse (1:2000, 7076S, Cell Signaling Technology). .. The following antibodies were used for immunoblotting: anti‐METTL3 (1:2000, ab195352, Abcam), anti‐METTL14 (1:3000, 26158‐1‐AP, Proteintech), anti‐WTAP(1:5000, 10200‐1‐AP, Proteintech), anti‐PRKN (1:1000, 14060‐1AP), anti‐p16 (1:1000, 10883‐1‐AP, Proteintech), anti‐p21 (1:1000, 2947S, CST), anti‐TRF1 (1:500, T1948, Sigma‐Aldrich), anti‐TRF2 (1:1000 dilution, 66893‐1‐IG, Proteintech), anti‐POT1 (1:500 dilution, 10581‐1‐AP, Proteintech), HRP‐conjugated goat anti‐rabbit (1:2000, 7074S, Cell Signaling Technology) or horse anti‐mouse (1:2000, 7076S, Cell Signaling Technology).
Article Title: PRKN-Mediated Ubiquitin-Proteasome Degradation of METTL3 Promotes Cellular Senescence.
Article Snippet: whole- cell lysates were incubated overnight with the appropriate antibodies, followed by incubation with Protein A/G beads (sc- 2003, SANTA CRUZ), or anti- Flag beads (Sigma). .. The following antibodies were used for immunoblotting: anti- METTL3 (1:2000, ab195352, Abcam), anti- METTL14 (1:3000, 26158- 1- AP, Proteintech), anti- WTAP(1:5000, 10200- 1- AP, Proteintech), anti- PRKN (1:1000, 14060- 1AP), anti- p16 (1:1000, 10883- 1- AP, Proteintech), anti- p21 (1:1000, 2947S, CST), anti- TRF1 (1:500, T1948, Sigma- Aldrich), anti- TRF2 (1:1000 dilution, 66893- 1- IG, Proteintech), anti- POT1 (1:500 dilution, 10581- 1- AP, Proteintech), HRP- conjugated goat anti- rabbit (1:2000, 7074S, Cell Signaling Technology) or horse anti- mouse (1:2000, 7076S, Cell Signaling Technology). .. The following antibodies were used for immunoblotting: anti- METTL3 (1:2000, ab195352, Abcam), anti- METTL14 (1:3000, 26158- 1- AP, Proteintech), anti- WTAP(1:5000, 10200- 1- AP, Proteintech), anti- PRKN (1:1000, 14060- 1AP), anti- p16 (1:1000, 10883- 1- AP, Proteintech), anti- p21 (1:1000, 2947S, CST), anti- TRF1 (1:500, T1948, Sigma- Aldrich), anti- TRF2 (1:1000 dilution, 66893- 1- IG, Proteintech), anti- POT1 (1:500 dilution, 10581- 1- AP, Proteintech), HRP- conjugated goat anti- rabbit (1:2000, 7074S, Cell Signaling Technology) or horse anti- mouse (1:2000, 7076S, Cell Signaling Technology).
Article Title: The PRKN/METTL3/CLDN2 axis promotes colorectal cancer development through epigenetic mechanisms.
Article Snippet: .. 2 mg of protein lysates were mixed with 1 μg of anti-Flag (rabbit pAb, Cat. No. 12076-1-AP, Proteintech), anti-PRKN (Cat. No. 14060-1-AP, Proteintech), anti-METTL3 (Cat. No. 15073-1-AP, Proteintech), or isotype IgG control (Cat. No. 30000-0-AP, Proteintech) antibody overnight at 4°C, which was followed by addition of Protein A/G agarose for 3 h. Protein in the immunoprecipitates was harvested for enrichment detection of ubiquitinated METTL3, METTL3 and PRKN by immunoblot analysis. .. Evaluation of mRNA and protein stability To evaluate the impact of METTL3 or IGF2BP2 on CLDN2 mRNA stability, transfected LoVo and HCT-116 cells were maintained for 24 h in media plus actinomycin D (Act D, 50 μg/mL, Selleck).
Control:Article Title: The PRKN/METTL3/CLDN2 axis promotes colorectal cancer development through epigenetic mechanisms.
Article Snippet: .. 2 mg of protein lysates were mixed with 1 μg of anti-Flag (rabbit pAb, Cat. No. 12076-1-AP, Proteintech), anti-PRKN (Cat. No. 14060-1-AP, Proteintech), anti-METTL3 (Cat. No. 15073-1-AP, Proteintech), or isotype IgG control (Cat. No. 30000-0-AP, Proteintech) antibody overnight at 4°C, which was followed by addition of Protein A/G agarose for 3 h. Protein in the immunoprecipitates was harvested for enrichment detection of ubiquitinated METTL3, METTL3 and PRKN by immunoblot analysis. .. Evaluation of mRNA and protein stability To evaluate the impact of METTL3 or IGF2BP2 on CLDN2 mRNA stability, transfected LoVo and HCT-116 cells were maintained for 24 h in media plus actinomycin D (Act D, 50 μg/mL, Selleck).
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