Fig 1: Production of Ph_Tat-CDKL5(1-352)-His M10V (45 kDa) (a), Ph_Sumo-Tat-CDKL5(1-352)-His M10V (57 kDa) (b), and Ec_Tat-CDKL5(1-352)-His (44 kDa) (c) in E. coli strains at 15 °C. (A) SDS-PAGE and (B) anti-His Western blot were carried out on total cellular extracts after 20 h of induction. NI, not-induced BL21(DE3); ArcticExp (DE3), ArcticExpress(DE3).
Fig 2: Preparation of Ph_Sumo-Tat-CDKL5(1-352)-His M10V, M10V_KD and wt variants. (A) SDS-PAGE of total extracts of BL21(DE3) after recombinant expression. NI, not-induced (B) IMAC elution fractions of Ph_Sumo-Tat-CDKL5(1-352)-His proteins. wt_dil, diluted wt. Arrows highlight a Ph_Sumo-Tat-CDKL5(1-352)-His wt fragmentation product due to an internal translation start.
Fig 3: Solubility of Ph_Sumo-CDKL5(1-352)-His M10V in the presence of E. coli molecular chaperones. After recombinant production for 22 h at 15 °C, soluble extracts were analyzed via SDS-PAGE. Tf, trigger factor; G, GroELS; G + Tf, GroELS + trigger factor; KJE, DnaK + DnaJ + GrpE; G + KJE, GroELS + DnaK + DnaJ + GrpE.
Fig 4: EB2 phosphorylation by Ph_Sumo-Tat-CDKL5(1-352)-His constructs. After the in vitro kinase assay, total EB2, and Ph_Sumo-Tat-CDKL5(1-352)-His variants were detected with an anti-His antibody (A), while phosphorylated EB2 was revealed by an anti-EB2 pSer222 antibody (B). q1\1\ a triplicate and contains Ph_Sumo-Tat-CDKL5(1-352)-His variants are in the following order: 1, KD; 2, M10V; 3, wt. (C) EB2 phosphorylation levels were detected at two time points and normalized for total EB2 and CDKL5ΔC levels. The results are reported as mean of triplicates and the error bars represent standard deviations. Asterisks indicate p < 0.05 in Student t-tests.
Fig 5: Effects of silent mutations on the internal translation start of CDKL5 encoding transcripts. (A) Modeling of the translation initiation rates from the M10-encoding ATG (underlined) in the CDKL5 transcripts used in this study. The initiation rate of the unmutated transcript was 195 au according to RBS Calculator [36], while the predicted effect of each substitution on such a parameter is indicated by blue numbers. (B) SDS-PAGE of total extracts of BL21(DE3) after recombinant expression of CDKL5ΔC variants. NI, not induced. (C) SDS-PAGE (left panel) and anti-flag Western blot (right panel) of P. haloplanktis TAC125 total extracts after recombinant expression of flCDKL5 variants. The arrows indicate the truncated form of CDKL5ΔC synthesized from M10.
Supplier Page from Abcam for Recombinant Human CDKL5 protein