PMC

Comparison of the capture efficiency between a conventional flat channel microfluidic chip and modified micropillar channel microfluidic chip (the microfluidic chip reaction chamber with an array of bypass columns defined as micropillar channel microfluidic chip) (n=4). Statistical significance was assessed by t-test. **P<0.01 vs. flat channel microfluidic chip.

(A): Pristine chip. (B) 24 h-nPDA decorated chip. (C): 5 min-sPDA decorated chip. (D): 10 min-sPDA decorated chip. (E): 20 min-sPDA decorated chip. (F): 30 min-sPDA decorated chip. (G): 1 h-sPDA decorated chip. (H): 2 h-sPDA decorated chip. (I): 4 h-sPDA decorated chip. (J): 8 h-sPDA decorated chip. All those samples had ultrasonically cleaned in distilled water for 10 min. Scale bar: 200 nm.

(A) Ube2w enhances CHIP’s ability to reduce cellular levels of a known CHIP substrate, INOS. Combinations of ^GFPINOS, CHIP and Ube2w were expressed in COS-7 cells by transfection. Coexpression of Ube2w enhances CHIP-mediated reduction of steady state levels of ^GFPINOS, whereas expression of Ube2w alone has no effect (n=3, *p < 0.05, **p < 0.005). Error bars indicate SEM. (B) RNAi knockdown of Ube2w increases levels of ^GFPINOS even when CHIP is overexpressed. HEK293 cells were transfected with combinations of ^GFPINOS, CHIP and vectors expressing shRNA against Ube2w or scrambled shRNA (n=3, *p < 0.05, **p < 0.005). Error bars indicate SEM. (C) In vitro ubiquitination of CHIP by Ube2w generates a single band corresponding to monoubiquitinated CHIP, which is absent in a control reaction lacking E1. Mass spectrometry identified K2 as the sole modified lysine on Ub-CHIP (see ). (D) The ubiquitinated lysine on CHIP, K2, is conserved in mammals but is absent in zebrafish and invertebrate species. The N-terminal sequences of CHIP orthologues were aligned using CLUSTAL-W (biology workbench); arrowhead indicates K2. (E) K2 is important for CHIP function in vivo. HEK293 cells were transfected to express ^GFPINOS alone or together with CHIP or CHIP^K2R. Coexpressed CHIP^K2R is less effective than wild type CHIP in decreasing steady state levels of ^GFPINOS (n=4, **p value <0.005, ***p value < 0.0005). Error bars indicate SEM.