Figure 2. Ub conjugation is not... | Rockefeller University Press

Figure 2.

$Figure 2. Ub conjugation is not necessary or sufficient for recruitment of GFP-DDFKBP to IBs. (A) Fusion to UbG76V decreases the half-life of folded Venus-DDFKBP. Cells were transiently transfected with UbG76V-Venus-DDFKBP or Venus-DDFKBP for 48 h in the presence of 1 µM shield-1, and mean Venus fluorescence of cells (n > 10) was determined by time-lapse microscopy after translation shutoff with 25 µM emetine. (B) Folded UbG76V-Venus-DDFKBP is constitutively ubiquitylated. Cells were transiently transfected with GFP-DDFKBP or UbG76V-Venus-DDFKBP in the presence of 1 µM shield-1 or vehicle for 72 h. Subsequently, Ub conjugates were affinity captured by incubating cell lysates with UBAhplic-2-conjugated beads, and eluate and unbound fractions were analyzed by SDS-PAGE and immunoblotting with anti-GFP and anti-Ub antibodies. (C) Folded and ubiquitylated UbG76V-Venus-DDFKBP is not recruited to IBs. Cells transiently transfected with httQ91-mCherry and UbG76V-Venus-DDFKBP for 48 h in the presence of 1 µM shield-1 were imaged by time-lapse microscopy. (D) E1 inhibitor blocks degradation of misfolded GFP-DDFKBP. U2-DDFKQ91 cells were treated with 1 µg/ml dox and 1 µM shield-1 for 48 h. Mean GFP-DDFKBP fluorescence of cells (n > 10) was determined by time-lapse microscopy after shield-1 washout in the presence or absence of 10 µM E1 inhibitor. (E) E1 inhibitor blocks ubiquitylation of misfolded GFP-DDFKBP. U2-DDFKQ91 cells incubated with 1 µM shield-1 were treated for 3 h with 10 µM E1 inhibitor or vehicle. After a 15-min shield-1 washout, Ub conjugates were affinity captured from cell lysates with UBAhplic-2 beads and eluate, and unbound fractions were analyzed by SDS-PAGE and immunoblotting with anti-GFP antibody. (F) Misfolded, nonubiquitylated GFP-DDFKBP is recruited to IBs. U2-DDFKQ91 cells were treated for 48 h with 1 µM shield-1 and 1 µg/ml dox. Cells were subsequently treated for 3 h with 10 µM E1 inhibitor, subjected to shield-1 washout, and imaged by time-lapse microscopy. (G) E1 inhibitor does not block recruitment of GFP-DDFKBP to IBs. GFP-DDFKBP fluorescence at IBs was measured in cells (n > 10) after shield-1 washout in the presence or absence of 10 µM E1 inhibitor. IB start indicates the frame in which httQ91-mCherry IBs were first detected. Time stamps indicate elapsed time in minutes. For C and F, t = 0 indicates the frame in which IBs were first observed, and negative values correspond to time in minutes preceding this frame. Data points indicate mean ± SD. Microscopy panels C and F are representative of n > 20 independent observations. Bars, 10 µm. Arrowheads indicate IBs. Western blots (WBs) are representative experiments from at least two independent repeats. Statistically relevant differences (α = 0.05): **, P < 0.0001.$

Ub conjugation is not necessary or sufficient for recruitment of GFP-DD_FKBP to IBs. (A) Fusion to Ub^G76V decreases the half-life of folded Venus-DD_FKBP. Cells were transiently transfected with Ub^G76V-Venus-DD_FKBP or Venus-DD_FKBP for 48 h in the presence of 1 µM shield-1, and mean Venus fluorescence of cells (n > 10) was determined by time-lapse microscopy after translation shutoff with 25 µM emetine. (B) Folded Ub^G76V-Venus-DD_FKBP is constitutively ubiquitylated. Cells were transiently transfected with GFP-DD_FKBP or Ub^G76V-Venus-DD_FKBP in the presence of 1 µM shield-1 or vehicle for 72 h. Subsequently, Ub conjugates were affinity captured by incubating cell lysates with UBA_hplic-2-conjugated beads, and eluate and unbound fractions were analyzed by SDS-PAGE and immunoblotting with anti-GFP and anti-Ub antibodies. (C) Folded and ubiquitylated Ub^G76V-Venus-DD_FKBP is not recruited to IBs. Cells transiently transfected with httQ91-mCherry and Ub^G76V-Venus-DD_FKBP for 48 h in the presence of 1 µM shield-1 were imaged by time-lapse microscopy. (D) E1 inhibitor blocks degradation of misfolded GFP-DD_FKBP. U2-DD^FKQ91 cells were treated with 1 µg/ml dox and 1 µM shield-1 for 48 h. Mean GFP-DD_FKBP fluorescence of cells (n > 10) was determined by time-lapse microscopy after shield-1 washout in the presence or absence of 10 µM E1 inhibitor. (E) E1 inhibitor blocks ubiquitylation of misfolded GFP-DD_FKBP. U2-DD^FKQ91 cells incubated with 1 µM shield-1 were treated for 3 h with 10 µM E1 inhibitor or vehicle. After a 15-min shield-1 washout, Ub conjugates were affinity captured from cell lysates with UBA_hplic-2 beads and eluate, and unbound fractions were analyzed by SDS-PAGE and immunoblotting with anti-GFP antibody. (F) Misfolded, nonubiquitylated GFP-DD_FKBP is recruited to IBs. U2-DD^FKQ91 cells were treated for 48 h with 1 µM shield-1 and 1 µg/ml dox. Cells were subsequently treated for 3 h with 10 µM E1 inhibitor, subjected to shield-1 washout, and imaged by time-lapse microscopy. (G) E1 inhibitor does not block recruitment of GFP-DD_FKBP to IBs. GFP-DD_FKBP fluorescence at IBs was measured in cells (n > 10) after shield-1 washout in the presence or absence of 10 µM E1 inhibitor. IB start indicates the frame in which httQ91-mCherry IBs were first detected. Time stamps indicate elapsed time in minutes. For C and F, t = 0 indicates the frame in which IBs were first observed, and negative values correspond to time in minutes preceding this frame. Data points indicate mean ± SD. Microscopy panels C and F are representative of n > 20 independent observations. Bars, 10 µm. Arrowheads indicate IBs. Western blots (WBs) are representative experiments from at least two independent repeats. Statistically relevant differences (α = 0.05): **, P < 0.0001.

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