5 tamra azide  (Jena Bioscience)

Journal: Nature

Article Title: Inhibitors supercharge kinase turnover through native proteolytic circuits

doi: 10.1038/s41586-025-09763-9

Figure Lengend Snippet: a , Expression proteomics of TAK285-treated NALM-6 cells. Kinases are highlighted where P < 0.05 (one-way ANOVA) and |log 2 [FC]| > 0.5. n = 3. b , Genome-wide FACS-based CRISPR–Cas9 screen. Essential genes, BLK, γ-secretase and proteasome subunits are highlighted in cases in which P < 0.05 (one-sided MAGeCK) and a log 2 [FC] > 2. n = 2. c , Flow cytometry assay for BLK stability reporter cells pretreated with DAPT followed by DMSO, TAK285 or HSP90i (6 h; NS, P = 0.9978 (DMSO), 0.9966 (HSP90i)). n = 3. d , Immunoprecipitation in BLK-Nluc-3×Flag K562 cells after pretreatment with alkynyl myristic acid (AMA, 100 µM), followed by 1 h carfilzomib (1 µM) and/or DAPT plus DMSO or TAK285. In-gel fluorescence (top, TAMRA) and Flag immunoblotting (bottom) analysis of the immunoprecipitated fractions. e , Stability reporter data for the indicated constructs treated with DMSO or TAK285 (left) (6 h; NS, P ≥ 0.9999 (FUS1), 0.9969 (SRC full length (FL))). n = 3. Right, FUS2 stability reporter data after pretreatment with DAPT (2 h) followed by DMSO or TAK285 (6 h; NS, P = 0.7333) n = 3. f , DMS data for the TAK285-treated (6 h) BLK stability reporter panel, displayed as the DMSO-normalized log 2 [FC] of sorted fractions. n = 3. g , Flow cytometry analysis of unique domain stability reporter fusions treated (6 h) with DMSO or TAK285. n = 3. pt., pretreatment. h , Flow cytometry analysis as in g for the indicated stability reporters. i , AlphaFold3-derived model of the BLK γ-secretase complex . Critical H-bonds are shown at the top right and the positioning of L3 is shown at the bottom right. Normalization of flow cytometry data was performed against the respective genotype/pretreatment unless specified otherwise. Data are mean ± s.d. n represents biological replicates. For c and e , statistical analysis was performed using two-way ANOVA with Tukey’s test for multiple comparisons. For a – h , inhibitor concentrations were as follows: TAK285 (2.5 µM), HSP90i (10 µM) and DAPT (12.5 µM).

Article Snippet: After removal of the supernatant, 56 μl of click-mix (170 μM TAMRA (5-TAMRA-Azide, CLK-FA008, Jena Biosciences), 230 μM copper sulfate, THPTA 1.15 mM, HCl 5 mM, sodium ascorbate 5 mM, in PBS) were added per sample.

Techniques: Expressing, Genome Wide, CRISPR, Flow Cytometry, Immunoprecipitation, Fluorescence, Western Blot, Construct, Derivative Assay

Journal: Nature

Article Title: Inhibitors supercharge kinase turnover through native proteolytic circuits

doi: 10.1038/s41586-025-09763-9

Figure Lengend Snippet: a Representative microscopy images for a BLK-GFP clonal cell line (RKO iCas9-BFP) assessed for different treatment conditions. Cells were pre-treated (pt.) for 1 h with TAK243 (1 µM) and DAPT (12.5 µM) followed by DMSO or TAK285 (2.5 µM) treatment (tr.) for the indicated timeframe. White arrows highlight localization patterns (n = 2 (clonal cell lines), m = 2). See SI Fig for additional example. b Quantified GFP intensity from images shown in (b) and additional timepoints. The mean total GFP abundance per cell was normalized per 0 h DMSO or TAK243 pre-treatment (n = 2 (clonal cell lines), m = 2). c Inhibition of myristoylation and measurement of BLK stability after TAK285 treatment. Myristoylation was inhibited 24 h prior to 6 h of 2.5 µM TAK285 treatment using the NMT1/2 inhibitor IMP-1088 (1 µM). Left: shown as DMSO-normalized data. Right: normalized per pre-treatment (x; DMSO or IMP-1088, respectively) (n = 3). d Representative microscopy images for localization of different BLK stability reporter pools (BFP-P2A-mCherry) in RKO iCas9 GFP cell line from Fig. and ( e ). e Stability reporter mutant panel of all phospho-susceptible residues in the unique domain of BLK, assessed after 6 h 2.5 µM TAK285 treatment using flow cytometry (n = 3). f Baseline stability values for BLK WT or BLK S6A stability KBM7 iCas9 reporter cells from ( e ) measured by flow cytometry (n = 3). g DMS data for IMP-1088 treated (1 µM, 24 h) BLK DMS stability reporter library depicted as normalized log 2 FC to DMSO (n = 3). h Orthogonal validation of selected mutations treated with either DMSO, TAK285 (6 h, 2.5 µM) or IMP-1088 (1 µM, 24 h) and analysed by flow cytometry (n = 3). i On bead TAMRA azide click for individual BLK mutants after FLAG immunoprecipitation (IP, n = 3). j Quantification of (i). k Representative microscopy images for individual BLK stability reporter mutations (n = 3). l Flow cytometric analysis of KBM7 iCas9 stability reporter SRC S3L, N4V cells pre-treated with DAPT, followed by DMSO or TAK285 (2.5 µM, 6 h) treatment. Data matched to Fig. (Two-way ANOVA with Tukey’s test for multiple comparisons, ns = 0.8754) (n = 3). All values represent the mean values ± SD; n = biological replicates, m = technical replicates. Scalebars = 25 µm.

Article Snippet: After removal of the supernatant, 56 μl of click-mix (170 μM TAMRA (5-TAMRA-Azide, CLK-FA008, Jena Biosciences), 230 μM copper sulfate, THPTA 1.15 mM, HCl 5 mM, sodium ascorbate 5 mM, in PBS) were added per sample.

Techniques: Microscopy, Inhibition, Mutagenesis, Flow Cytometry, Biomarker Discovery, Immunoprecipitation