Anti-Ubiquitin 抗体 (ab19247)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes.  ab19247 and ab8245 (loading control to GAPDH) were diluted 1/200  and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging using the Licor Odyssey CLx.

Scrambled and lentivirus-stable infected SN4741 cells grown on coverslips were induced to differentiate under restrictive conditions for 24 h (pre-lethal stage), fixed and permeabilized with 0.1% Triton X-100 + 0.1% sodium citrate for 5 minutes. Cells were blocked with 10% donkey serum in PBS for 1 hour at 37°C and incubated over night at 4°C with rabbit polyclonal antibody against ubiquitin (ab19247, 1:100) and mouse monoclonal α-synuclein antibody (1:25). Samples were washed twice with PBS for 10 minutes before the addition of secondary antibodies at room temperature in the dark for 1 hour

Scrambled infected (control) cells show a diffuse staining pattern and do not show any inclusions at both permissive and restrictive temperatures (upper images). SKP1A-deficient cells developed multiple and perinuclear (bottom images, see arrows and insets) inclusion bodies 24 h after induction of differentiation, with characteristics of aggresomes. The reactivity of the aggregates to the different antibodies demonstrated a similar pattern as indicated by co-localized yellow immunostaining in the overlaid right-hand panel. No fluorescence was detected when the primary antibody was omitted. Each panel shows a representative picture of 10–15 views in two independent experiments.

Images top - bottom:

Scrambled 33°C/10% FCS,

SKP1A shRNA-1 33°C/10% FCS,

Scrambled 39°C/0.5% FCS, 

SKP1A shRNA-1 39°C/0.5% FCS.