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Fig. 5 | Acta Neuropathologica Communications

Fig. 5

From: C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease

Fig. 5

Expression of C9orf72 and SMCR8 proteins are positively correlated in cell lines and human brain tissues. a C9orf72-FL was coexpressed in HEK 293T cells with 3 different epitope-tagged SMCR8 constructs, FLAG-tagged RO60 protein, or empty vectors (pcDNA3 and pcDNA6 myc/his B). A Western blot of whole cell lysates was probed sequentially with rb α-FLAG, ms α-HA, ms α-V5, and rb α-HSP90 antibodies, the latter as a loading control. At the exposure time for the film shown, expression of C9orf72-FL was not seen in the presence of empty vector or RO60-FL, but signal was robust in the presence of SMCR8. b Western blot of brain motor cortex tissue lysates of C9ALS patients (lanes 1–5) and unaffected control individuals (lanes 6–9) probed with α-SMCR8 and α-HSP90 antibodies. Sample names are shown above the panels (see Table S4). Numbers below the middle panel are normalized ratios of SMCR8 to HSP90 expression determined by ImageJ analysis of band intensities and calculated as described in the text. The lower panel shows the approximately 150-kD unspecified band detected by α-WDR41-SC antibody in human brain tissue lysates (see Fig. S1F): this panel is included only as an additional loading control and is not intended to show expression of canonical WDR41 protein. Approximtely 50 μg of protein was loaded in each lane. c Dot plot of ratios of SMCR8 to HSP90 protein band intensities determined by ImageJ analyses of brain tissues lysates from 11 C9ALS and 10 control individuals. Each sample point is the average of 2 to 4 independent Western blot analyses. A short horizontal line indicates mean values. The presence of a C9orf72 hexanucleotide expansion in each C9ALS carrier individual was confirmed by Columbia University and Target ALS using RP-PCR and Illumina Expansion Hunter, but expansion copy numbers are not known

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