The Journal of Neuroscience, September 27, 2006, ():
RTP801 Is Elevated in Parkinson Brain Substantia Nigral Neurons and Mediates Death in Cellular Models of Parkinson's Disease by a Mechanism Involving Mammalian Target of Rapamycin Inactivation
J. Neurosci. Malagelada et al.
26: 9996
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Supplementary data.
(A) Detection of RTP801 with the antibody against human protein (Chemicon, CA.): Loss of signal following knockdown with shRNA. Human Embryonic Kidney (HEK) 293 cells were transfected with pSIREN-shLuciferase, pSIREN-shRTP801B, or pSIREN-shRTP801C. 24 hours later, cell extracts were collected for each condition and levels of endogenous RTP801 protein were analyzed by western immunoblotting. When endogenous RTP801 was knocked down with shRTP801B, which is specific for the human RTP801 sequence, there was an evident decrease in the band (at apparent MW 33 Kda) corresponding to RTP801.. In contrast, with shRTP801C, which targets the mouse and rat sequence but not human, we did not observe any change in the RTP801 band relative to that in control or shLuc samples. The membrane was reproved with an antiserum against ERK1 to establish equal loading and with an antibody against DsRed, the co-expressed fluorophore, to indicate relative transfection efficiency.
(B) Detection of murine RTP801 with the antibody against RTP801 (Santa Cruz, CA.): Loss of signal following knockdown with shRNA. Murine CAD cells were transfected with pSIREN-shLuciferase, as a control or with pSIREN-shRTP801A or pSIREN-shRTP801C. 24 hours later, cell extracts were collected and levels of endogenous RTP801 protein were analyzed by western immunoblotting. When endogenous RTP801 was knocked down either shRTP801A or shRTP801C, both of which are specific for the rat and mouse RTP801 sequence, we observed an evident decrease in the band corresponding to RTP801. The membrane was reproved with an antiserum against ERK1 to establish equal loading and with an antibody against DsRed, the co-expressed fluorophore, to indicate relative transfection efficiency.
