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P23 as loading control western blot8/11/2023 ![]() ![]() Each lane contained 20 mug of whole cell extract. After incubation at 37degC for 60 h, the cells were harvested and whole cell extract was prepared for Western analysis. Cells (3x10 6 in 0.4 ml) were transfected with either pcDNA-V5 or pcDNA-V5-p23 plasmid (30 mug in 30 mul) by electroporation (190 V, 70 ms). C, Western results showing that V5-p23 restored the AhR levels in p23kd1-2 cells. The images (above) are a representation of the triplicate data. The plot (bottom) is the quantified data showing the means with error bars (mean +- SD, n = 3). Each lane contained 19-27 mug of whole cell extract and was normalized by beta-actin. Protein levels in WT are arbitrarily set as 1. Western results showing p23 (A) and AhR (B) levels. Five cell lines examined: wild type (WT), negative control knockdown stable (NC), and p23 knockdown stable p23kd1-3. 1 p23 and AhR protein levels in p23-specific knockdown stable Hepa1c1c7 cells generated by electroporation. Results are representative of at least three independent experiments.įig. The indicated proteins were incubated with recombinant active caspase-3 for 2 h at 37 degC. C, caspase-3-mediated processing of the purified recombinant proteins over the indicated concentration range. The indicated proteins were incubated with recombinant active caspase-1 for 2 h at 37 degC. B, caspase-1-mediated processing of the purified recombinant proteins over the indicated concentration ( Conc. Substrate proteolysis was detected by SDS-PAGE followed by immunoblotting. S1 ) as described under ""Experimental Procedures."" Recombinant IL-1beta (10 ng per reaction) was added to cell-free extracts as a positive control for caspase-1 activity. Recombinant caspases were active site titrated (see supplemental Fig. A, processing of substrate proteins in Jurkat cell-free extracts after addition of the indicated amounts of recombinant caspase ( Casp )-1, -3, and -7 followed by incubation for 2 h at 37 degC. Caspase-1 exhibits greater promiscuity toward natural substrates than caspases-3 or -7. (F) CRISPR-treated mice had paw incision surgery performed on day 9, with injection of 3.2 mg/kg moįIGURE 4. CRISPR treatment reduced p23 signal by 36.3% and Cdc37 by 46.0%. same target Negative Control group by Unpaired 2-Tailed t -test. All mice treated in one technical replicate, with staining and analysis of the resulting tissue performed in more than one technical replicate. (E) Data from (D) for all mice quantitated as described in the ""Materials and Methods"" section. Both targets (p23 - red, Cdc37 - green) have a similar staining pattern to Hsp90alpha, and both are clearly reduced by CRISPR treatment. Representative images shown from the PRN. (D) p23, Cdc37, or Negative Control CRISPR-treated mice with icv delivery of constructs analyzed by IHC for target knockdown on day 10. same time point inhibitor treatment group by two-way ANOVA with Fisher's Least Significant Difference post hoc test. Experiments performed with two technical replicates for each drug. (A-C) Ten nanomoles of Gedunin (p23, A ), 10 nmol of Celastrol (Cdc37, B ), or 0.1 nmol of KU177 (Aha1, C ) or Vehicle control injected icv concurrently with paw incision surgery with a 24 h recovery, followed by 3.2 mg/kg morphine sc. Male CD-1 mice used for all experiments, data reported as the mean +- SEM, with sample sizes of mice/group noted in each graph. ![]() The images were captured at 60x magnification.įigure 4 Identification of the co-chaperones p23 and Cdc37 as promoters of opioid anti-nociception in the brain. Panel e represents control cells with no primary antibody to assess background. Panel d represents the merged image showing Nucleus and cytoplasmic localization. F-actin (Panel c: Blue) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). The cells were labeled with p23 Monoclonal Antibody (JJ3) (Product # MA3-414) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Product # A32787), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. Immunofluorescence analysis of p23 was performed using 70% confluent log phase HeLa cells. ![]()
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