29-13 DHH1 RNAi uninduced 29-13 DHH1 RNAi induced 29-13 SCD6 RNAi uninduced 29-13 SCD6 RNAi induced DHH1/eYFP-DHH1 SCD6/SCD6-eYFP wild type Supplementary Figure 3A 175 83 eYFP-DHH1 62 47.5 32.5 25 16.5 anti-DHH1 175 83 SCD6-eYFP 62 47.5 32.5 25 16.5 anti-SCD6 175 83 eYFP-DHH1 SCD6-eYFP 62 47.5 32.5 25 anti-eYFP anti-BiP Supplementary Figure S3 B eYFP-DHH1 in culture after washes after fixation SCD6-eYFP in culture after washes after fixation Supplementary Fig. S3 C anti-DHH1 DHH1 RNAi uninduced DHH1 RNAi induced eYFP-DHH1 wild type 8 8 2 2 anti-SCD6 SCD6 RNAi uninduced SCD6 RNAi induced SCD6-eYFP wild type 2 2 2 2 anti-eYFP eYFP-DHH1 SCD6-eYFP wild type 2 2 2 exposure time (s) exposure time (s) exposure time (s) Supplementary Fig. S3 D eYFP-DHH1 SCD6-eYFP eYFP-DHH1 SCD6-eYFP anti-DHH1 anti-SCD6 anti-GFP anti-GFP merge merge merge merge supplementary Figure 3 Fig. S3: Comparison of the determination of the subcellular localisation of P-body components by immunofluorescence and fluorescent protein fusions. The following cell lines were used: Lister 427 Lister 427 expressing eYFP-DHH1 from the endogenous locus Lister 427 expressing SCD6-eYFP from the endogenous locus Lister 427:pLEW13:pLEW29 with an integrated p2T7-177-derived DHH1 RNAi construct Lister 427:pLEW13:pLEW29 with an integrated p2T7-177-derived SCD6 RNAi construct A) Monospecificity of antibodies Western blot of the cell lines above probed with anti-DHH1 or anti-SCD6 or anti-eYFP. The arrows indicate the reduced signal observed after the induction of RNAi. In cell lines expressing eYFP fusion proteins, the expression from the transgene was less than the expression from the remaining unaltered locus. B) Fixation has no effect on the subcellular localisation of eYFP-DHH1 and SCD6-eYFP. All images are of the fluorescent protein. All cell washes and fixations were performed in SDM-79 medium without haemin or serum. C) Specificity of antibodies in immunofluorescence experiments For anti-DHH1 and anti-SCD6 the signal was compared for an RNAi cell line before and after induction of RNAi. For anti-eYFP the cell lines expressing eYFP-DHH1 and SCD6-eYFP were compared with the parental cell line. Standard immunofluorescence procedures were used. All cell washes and fixations were performed in SDM-79 medium without haemin or serum. Prior to the experiments paraformaldehyde concentration and time of fixation were titred from 2% paraformaldehyde for 5 minutes to 4% paraformaldehyde for 60 minutes. The final fixation was 3% paraformaldehyde for 30 minutes. These values were chosen for retention of morphology and fluorescent protein signal and strength of antibody signal. Post-fixation permeablisation with either 0.1% triton TX-100 or methanol at -20oC were tested. The use of methanol resulted in the loss of eYFP fluorescence and did not alter the antibody staining appreciably, 0.1% triton TX-100 was used in all subsequent experiments.The results show that all antibody signals obtained by immunofluorescence were specific. D) Comparison of subcellular localisation determined by eYFP fluorescence and antibody staining. Anti-DHH1 and anti-eYFP were used to stain cells expressing eYFP-DHH1 and anti-SCD6 and anti-eYFP were used to stain cells expressing SCD6-eYFP. In all cases the antibody fluorescence and eYFP fluorescence overlapped but were not identical. The eYFP fluorescence was concentrated in discrete foci in the cytoplasm, which were readily visible against the more diffuse staining in the cytoplasm. In contrast, the foci had only slightly stronger fluorescence than the cytoplasm in antibody staining experiments. The absence of precise co-incident distribution of anti-eYFP and the eYFP fluorescence is most readily explained by the failure of antibodies to penetrate P-bodies after fixation so that the foci are visualised by surface binding of antibodies but through fluorescence of the whole structure when fluorescent proteins were used.. Thus, the relative fluorescence of the foci compared to the cytoplasm is greater with eYFP fluorescence which originates throughout the P-body, than with antibody fluorescence which only stains the surface of the P-body.
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