Autoradiogram of the SDS-PAGE analysis in (B). multiple bands, with no evidence for any specific acknowledgement of ZC3H11. There was a very strong transmission at about 50 kDathe same position as alpha and beta tubulin. Cytoskeleton-free extracts (lanes 5C8) were therefore obtained as explained in Materials and Methods. The putative ZC3H11 protein band is usually indicated with an arrow (lane 6). This is present only in heat-shocked cells without RNAi. A cross-reacting band (probably residual tubulin) is usually indicated by an asterisk. After more careful fractionation this band is usually not seen, as judged by signals at 27C (Fig 1A) and controls with RNAi (Fig 2A, lane 15). C. Pull-down with anti-ZC3H11 antibodies. Extracts from 5107 procyclic cells ectopically expressing full-length or N-terminal fragment of myc-tagged ZC3H11 were subjected to immunoprecipitation with anti-ZC3H11. The efficiency of immunoprecipitation was analysed by Western blotting using anti-myc. In: input, U: unbound (2106 cell-equivalents), E: eluate (5106 cell-equivalents). D. Western blot analysis of ZC3H11-myc obtained by cell fractionation. Cytoskeleton-free extracts from JAK3 covalent inhibitor-1 control and heat-shocked procyclic cells ectopically-expressing ZC3H11-myc protein were analyzed by immunoblot with anti-myc and, as control, anti-aldolase. Approximately 90% of ectopically-expressed ZC3H11-myc protein remained in the supernatant after cytoskeleton depletion (S1D Fig). E. Expression of endogenous ZC3H11 in procyclic cells after warmth shock. Cells were treated for 1 hour with moderate (37C) or severe (41C) heat shock. To the cytoskeleton-free extracts from 5106 cells were added dilutions of recombinant ZC3H11 fragment and analyzed by immunoblotting. Detection was with anti-ZC3H11 and cross- reacting band is shown as loading control. One representative image from three individual experiments is shown. F. ZC3H11 protein levels in procyclic cells with normal and warmth shock conditions. Quantification from JAK3 covalent inhibitor-1 three imitation immunoblots is shown. We cannot exclude some cross-reactivity in the transmission for 27C. G. Dynamics of ZC3H11 expression in procyclic forms during moderate heat shock and a recovery period. The cells were incubated for 1h at 37C, and then transferred to normal conditions. The loading control is one of the major cross-reacting bands seen in panel B. ZC3H11 accumulation was visible within 5 minutes. Loss of ZC3H11 at 27C was slower: it started to decrease only after an hour, but was notably less abundant after 5h.(PDF) ppat.1005514.s002.pdf (2.8M) GUID:?4DD9F382-951D-4B34-A29D-6B0437BFD759 S2 Fig: Negative results. A. Stresses that do not induce ZC3H11 expression in procyclic forms. All treatments were for 1h and ZC3H11 was detected HSPA1 in cytoskeleton-free extracts using the polyclonal antibody. A cross-reacting band served as a loading control. B. RNAi targeting protein kinases Tb927.10.5140 and Tb927.5.2820 has no effect on ZC3H11 band migration. RNAi was induced for 2 days, then the cells were transferred to either 37C or 41C for 1 hour. ZC3H11 JAK3 covalent inhibitor-1 was detected by western blotting, with a cross-reacting band that is unaffected by heat shock as loading control. Cytoskeleton-free extracts from 5106 cells were loaded on each lane. One representative image from three separate experiments is shown. C. Effect of RNAi targeting DRBD2, DRBD7, PUF3, RBP9, RBP31, ZC3H8, ZC3H13, ZC3H22, JAK3 covalent inhibitor-1 ZC3H32, ZC3H35, ZC3H39, 4E-IP and Tb927.11.14220 on ZC3H11 expression in bloodstream and procyclic trypanosomes. Procyclic RNAi cell lines targeting DRBD2, ZC3H39, ZC3H22, 4E-IP and bloodstream form RNAi cell lines targeting DRBD7, JAK3 covalent inhibitor-1 PUF3, RBP9, RBP31, ZC3H8, ZC3H13, ZC3H32, ZC3H35, Tb927.11.14220 were induced with tetracycline (200g/ml) for 2 days. The unstressed and heat-shocked parental cell lines were used as a control. ZC3H11 levels were analysed by Western blotting. 5106 cells were loaded per lane. A cross-reacting band served as a loading control.(PDF) ppat.1005514.s003.pdf (2.6M) GUID:?348955EA-6855-4B6D-B23A-23104583DF9F S3 Fig: A. mRNA migrates near the 40S peak in 10C30% sucrose gradients. The upper panel shows absorbance at 254nm after 10C30% sucrose density gradient centrifugation of extracts from procyclic trypanosomes grown at 27C. Peaks for small (40S), monosome (80S) and polysomes are indicated with arrows. The lower panels show the corresponding methylene blue stain (for rRNA) and Northern blot detection of mRNA. B. Cycloheximide treatment does not affect mRNA migration in sucrose gradients. Extracts from untreated or cycloheximide treated procyclic cultures grown at 27C were subjected to 17.5C50% sucrose gradient centrifugation. The upper panels show representative absorbance profiles at 254 nm, and the lower panels are Northern blots of RNA preparations from the different fractions. A methylene blue stain is shown beneath the fraction numbers. mRNA was detected in both blots. C. Cutting of mRNA with RNase H moves the fragments into a fraction above the 40S peak. Procyclic.