kdm2a/fbxl11 antibody  (Bio-Techne corporation)

Journal: International journal of molecular sciences

Article Title: KDM2A and KDM3B as Potential Targets for the Rescue of F508del-CFTR.

doi: 10.3390/ijms23179612

Figure Lengend Snippet: Figure 3. Transcriptional and stability analysis of F508del-CFTR after demethylases downregulation. (A) CFTR mRNA levels determined by quantitative real-time PCR in F508del-CFTR expressing CFBE41o- cells were transfected with two different siRNAs for each target (JMJD6, KDM2A, KDM3B) for 48 h. CFTR mRNA expression was normalized to 18S RNA and reported relative to its expression in control (SCR) cells that was arbitrarily set to 1 (means ± SD values, n = 5). (B) F508del-CFTR overexpressing CFBE41o- cells were transfected with siRNA targeting JMJD6, KDM2A, KDM3B or with scrambled siRNAs for 48 h. Subsequently, protein synthesis was inhibited by adding 100 µg/mL cycloheximide (CHX) and cells were harvested after 30, 60 and 90 min. Protein lysates were analyzed by western blot with an anti-CFTR antibody. Calnexin was used as a loading control. The lower-right graph represents the densitometric quantification of the immunostained bands of F508del-CFTR band B normalized by the value at time = 0 (mean ± SD, n = 4; * p < 0.05 vs. the value of the same time-point of SCR).

Article Snippet: Anti-KDM2A (FBXL11) (NB100-74602) antibodies were from Novus Biological (Centennial, CO, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Control, Western Blot

Journal: International journal of molecular sciences

Article Title: KDM2A and KDM3B as Potential Targets for the Rescue of F508del-CFTR.

doi: 10.3390/ijms23179612

Figure Lengend Snippet: Figure 4. Mutational analysis of CFTR methylated lysine and effect of demethylases downregulation on chaperone machinery. (A) CFBE41o- cells were transiently transfected with F508del-CFTR, or other derivative mutants as indicated. After 24 h, cells were treated with 3 µM VX-809 and the amounts of F508del-CFTR band B and band C were assessed by western blotting. α-tubulin was used as loading control (upper panel). In the lower panel, the graphs show the ratio of F508del-CFTR band C/band B for the cells treated with VX-809, obtained by the densitometric quantification of the immunostained band C normalized by band B expression, and expressed as a percentage of the control cells (Ctrl) (means ± SD values, n = 4; * p < 0.05 vs. Ctrl). (B) F508del-CFTR expressing CFBE41o- cells were transfected with siRNA (#1 and #2 guides) targeting KDM2A or KDM3B or with a scrambled (SCR) siRNA for 48 h. In the left and central panels, the expression of the indicated chaperones was analysed by western blotting. α-tubulin was used as loading control. In the right panel, the graphs report the densitometric quantification of the immunostained bands of the experiment detailed in the left and central panels normalized by α-tubulin expression, and expressed as a percentage of the control cells (SCR). (Means ± SD values, n = 3; * p < 0.05 vs. SCR).

Article Snippet: Anti-KDM2A (FBXL11) (NB100-74602) antibodies were from Novus Biological (Centennial, CO, USA).

Techniques: Methylation, Transfection, Western Blot, Control, Expressing