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?2a and b). To more accurately determine whether T185 and/or S185 TMEM106B levels differently alter endogenous PGRN levels, we harvested T185- and S185-transfected HeLa cells 2?days after transfection and quantified both intra- and extracellular levels of PGRN using a PGRN ELISA. TMEM106B over-expression of either isoform caused a significant increase in PGRN levels in both intracellular and in the media as compared with control-transfected #links# cells (Fig.?2c). These results confirm earlier observations published by Brady et?al. and indicate that a direct effect on PGRN levels is unlikely to explain the risk associated with p.T185S in TMEM106B (Brady et?al. 2013). Upon over-expression of T185 and S185 #links# TMEM106B as part of our PGRN-related studies, we consistently observed a more prominent TMEM106B-immunoreactive band in T185-transfected cells (Fig.?2d). Upon quantification of TMEM106B protein levels, S185 expression was approximately 37% that of T185 expression both 2 and 3?days post transfection (p?<?0.0001) (Fig.?3a and b). To determine whether these differences in TMEM106B isoform expression were because of changes at the RNA level, TMEM106B RNA levels were measured in T185- and S185-transfected cells at the same time points. Quantitative PCR analyses confirmed that TMEM106B RNA levels are not different in T185 versus S185 over-expressing cells at 2 or 3?days (Fig.?3c and d). To eliminate the possibility that differences between T185 and S185 protein expression are plasmid-specific, we further cloned the TMEM106B isoforms into a pAG3 mammalian expression plasmid and transfected these constructs into HeLa cells for 3?days. Consistent with our pAAV TMEM106B constructs, S185 TMEM106B protein was significantly less expressed than T185 TMEM106B (56% of T185 levels; p?<?0.05), independent of RNA levels (Figure S3a, c, e). To ensure that these observations were not specific only to HeLa cells, we also transformed #links# T185 and S185 TMEM106B into human embryonic kidney (HEK-293T) cells for 3?days, after which protein and RNA levels were quantified. Similar to HeLa cells, HEK-293T cells also expressed S185 TMEM106B significantly less than T185 (55% of T185 levels; p?<?0.05), independent of RNA levels (Figure S3b, d, f). Taken together, these results suggest that a post-translational mechanism is responsible for the differences in protein levels between the risk (T185) and protective (S185) isoforms of TMEM106B. To determine whether T185 and S185 TMEM106B proteins are degraded at different rates, we treated T185- and S185-transfected cells with 20?��g/mL of cycloheximide to block protein synthesis.</p>