Fig. 5: UA interacting with MLCK diminished its degradation.

A Schematic diagram of cellular thermal shift assay (CTSA) in CCSMC pre-treated by HUA, then equiparting and heating with 3 °C gradient temperature from 37 to 64 °C. The soluble MLCK levels in the supernatant were detected by WB. B Representative WB images of remaining MLCK levels after heating in CCSMC supernatant detected by CTSA. C Protein quantification of the melting point where 50% MLCK remained from (B). D, E Uric acid structure chart and the schematic diagram of the amino acid from MLCK for UA-interacting. F Representative WB images showing remaining MLCK after heating in the supernatant of ARPE19 cells transfected with plasmid overexpressing WT MLCK, N803A/K799A mutated MLCK (MLCK-WT, N803A, and K799A) for 48 h. Heating with 3 °C gradient temperature from 37 to 58 °C among the range in (B). G Protein quantification of the melting point where 50% MLCK remained in (F). H Representative WB images showing remaining MLCK after heating in the supernatant of CCSMC transfected with plasmid overexpressing control, WT MLCK, N803A/K799A mutated MLCK (Vector, MLCK-WT, N803A, K799A) for 48 h under HUA condition or not. Heating with 3°C gradient temperature from 40 to 55 °C among the range in (B). I Protein quantification of the melting point where 50% MLCK remained in (H). J Representative WB images of MLCK protein levels collected at 0, 4, 8, or 12 h after CHX or CHX + MG132 treatment in CCMC under HUA condition or not. K The line chart of the relative MLCK protein levels from (J). (L) Table showing the P values of (J) in different groups and hours analyzed by the two-tailed unpaired t-test. CHX = 50 μM. MG132 = 50 μM. n = 3 independent biological replicates. HUA means 10% DMEM with an extra 450 μM uric acid for 48 h after 4 h starvation.