Fig 1: Immunofluorescence microscopy shows evidence for association of endogenous SMCR8 protein with cytoplasmic aggregates. a FLAG-tagged SMCR8 and RFP-tagged ubiquitin transfected in 2102Ep cells colocalize in a structure consistent with the aggresome. b Overexpression of V5-tagged C9orf72 does not induce stress granule formation in unstressed U2OS cells. c Exogenously expressed HA-SMCR8 protein is not observed in SGs of U2OS cells stressed with NaAsO2. d WDR41-FL protein does not colocalize with SG marker protein TIA1 in U2OS cells stressed with NaAsO2. e Endogenous C9orf72 protein detercted by the a-SMCR8-SC antibody does not colocalize with SGs in NaAsO2-stressed U2OS cells (see also Fig. S4E). f Endogenous C9orf72 protein detected by the C9-L antibody [54] does not colocalize with SGs in DTT-stressed U2OS cells. g,h Endogenous SMCR8 detected by the a-SMCR8-ab202283 antibody localizes to SGs of stressed (h), but not unstressed (g) U2OS cells (see also Fig. S4G-I). i The a-WDR41-SC antibody does not detect endogenous protein in SGs of NaAsO2-stressed 2102Ep cells. NT: no treatment. Cell nuclei were stained with Hoechst 33342 (right-most panels). Size bars are 10 µm
Fig 2: 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 a-FLAG, ms a-HA, ms a-V5, and rb a-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 a-SMCR8 and a-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 a-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
Fig 3: Protein interaction analyses by Western blotting and co-IP of the SMCR8 complex in HEK 293T cells (see Fig. S1 for antibody analyses). a Endogenous C9-L (arrow) co-IPs with FLAG-tagged SMCR8. The thick arrowhead marks a band consistent in size with C9-S. b FLAG-tagged C9orf72 co-IPs both endogenous and co-transfected HA-tagged SMCR8. c FLAG-tagged WDR41 protein co-IPs both endogenous C9orf72 and SMCR8 proteins (indicated by arrows). Tagged C9orf72 and WDR41 proteins of (b) and (c) are not visible in whole cell lysates at the Western blot film exposure times shown. d C9orf72-FL, FL-SMCR8, and empty vector were immunoprecipitated on a-FLAG agarose from transfected 293T whole cell lysates, resolved on a polyacrylamide gel, and silver-stained. IP reactions were in the presence or absence of 50 µg/ml RNases. Complex immunoprecipitate samples were analyzed by MS sequencing. Arrows indicate full-length protein bands. Protein molecular weight markers are those of Novex Sharp Pre-stained Protein Standard (Thermo Fisher Scientific)
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