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. 2016 Aug 11;166(4):935–949. doi: 10.1016/j.cell.2016.07.001

Figure 2.

Figure 2

Heat Stress Activates UBQLN2 to Clear Aggregated Proteins

(A) UBQLN2 depletion by siRNA leads to defective clearance of heat-shock-induced insoluble ubiquitin conjugates (left), and quantification of insoluble ubiquitin in the pellet (right) (n = 2). Error bars represent SEM.

(B) Insoluble heat-shock-generated ubiquitin conjugates are cleared efficiently in ATG5 knockout (autophagy-deficient) MEFs in a proteasome-dependent manner.

(C) UBQLN2 depletion in autophagy-deficient cells leads to attenuated clearance of heat-shock-induced insoluble ubiquitin conjugates. Quantification (n = 3) is shown (right). Error bars represent SD; statistical tests were two-tailed t tests.

(D) HSP70 siRNA leads to a defective clearance of ubiquitylated aggregated proteins. Over time, the transcriptional heat shock response leads to increased levels of HSP70.

(E) Increased interaction of UBQLN2 with HSP70 and ubiquitin was observed in HSP105 knockout (KO) MEF cells.

(F) UBQLN2 and ubiquitin are more abundant in the pellet fraction after heat shock in HSP105 KO MEF cells.

(G) HSP105 KO MEFs are deficient in clearing heat-shock-induced aggregates. In addition, increased binding of HSP70 and ubiquitin to UBQLN2 was detected.

(H) Depletion of UBQLN2 by siRNA leads to defective clearance of puromycin-labeled truncated proteins.

(I and J) UBQLN2 translocates to the nucleus after heat stress (see Figure S2A for fractionation protocol). Quantification of the normalized nuclear fluorescence intensity is shown (J, bottom) (n = 99 and 122 for 37°C and 43°C, respectively). Error bars represent SD.

(K) UBQLN2 co-localizes with cellular HTT aggregates in HEK293 cells inducibly expressing pathological GFP-Huntingtin (HTTQ103).

(L) UBQLN2 co-aggregates with pathological, but not non-pathological, GFP-Huntingtin, as shown by filter trap assay.

(M) UBQLN2 depletion leads to increased HTT-Q103 aggregates, running in the stacking gel. Quadruplicate transfections are shown.

See also Figures S2, S3, S4, and S7.