Figure 1.
2D-DIGE sample preparation flowchart & preparatory gel image. A.
Schematic diagram of crucial steps in 2D-DIGE sample preparation. B. Preparatory Gel image showing differentially expressed protein spots. The spots are circled along with their corresponding spot numbers (Master/Spot IDs).
Figure 2.
Differential expression of ATP synthase subunit β, inorganic pyrophosphatase and calmodulin.
A and B shows images of ATP synthase subunit β (spot no. 737) in control and 3 hpi or 9 hpi RV infected samples respectively. D and E shows images of Inorganic Pyrophosphatase (spot no. 1273) in control and 3 hpi or 9 hpi RV infected samples respectively. G and H shows images of Calmodulin (spot no. 1643) in control and 3 hpi or 9 hpi RV infected samples respectively. C, F and I show the Standard Log Abundance plot in standard, control and treated sets of all replicate gels respectively for ATP synthase subunit β, inorganic pyrophosphatase and calmodulin.
Table 1.
List of differentially modulated proteins during GARV SA11 infection in HT-29 cells.
Figure 3.
Validation of differentially regulated proteins by immunoblotting and qRT-PCR.
A & B. Protein were extracted and loaded onto SDS-PAGE from HT 29 cells infected with GARV SA11 strain for indicated time periods followed by Western Blotting. Results shown here are representative of triplicated immunoblotting experiments. C & D. 2 µg RNA of SA11 infected (at various time points) HT29 cells were converted to cDNA. 1 µl of each sample were taken for Real-time PCR and amplified with different primers in triplicates. Average value of relative fold change as expressed by 2−ΔΔCt were plotted for each time points. mRNA level of Superoxide dismutase (SODC), elongation factor 1-β (EF1B), peroxiredoxin-6 (PRDX6) and RV-NSP4 are analyzed here.
Table 2.
Comparison of fold change in 2D-DIGE and Western Blotting of cellular proteins.
Figure 4.
Cellular localization and colocalization pattern of CaM in confocal microscopy.
HT29 cells infected for 0 hpi, 3 hpi and 9 hpi were fixed with paraformaldehyde and incubated with CaM and RV VP6 antibody, followed by FITC labeled mouse and Rhodamine labeled rabbit antibodies. High amount of CaM (green) expression was observed at 3 hpi compared to 0 hpi and 9 hpi. Amount of VP6 (red) was higher at 9 hpi than 3 hpi with no expression at 0 hpi. CaM was colocalized with VP6 as evidenced by merged confocal image.
Figure 5.
Modulation of CaM in infection of various RV strains. A.
Expression of cellular CaM was investigated in infection of Wa, NCDV and OSU strains of RV by immunoblotting at 0 hpi, 3 hpi & 9 hpi. B. CaM mRNA level in infection of SA11-H96, Wa, OSU and NCDV strains of RV were analyzed at 3 hpi & 9 hpi.
Figure 6.
Validation of modulated proteins in in vivo model. A.
Confirmation of RV infection in mice by qRT-PCR of NSP4 mRNA. B. Validation of differentially regulated Proteins in in-vivo model. Proteins were extracted from the BALB/c mice infected with GARV SA11 in ligated intestine, 100 µg of protein was loaded onto SDS-PAGE followed by immunoblotting. Results shown here are representative of triplicate experiments. Relative band intensity with respect to GAPDH is expressed as bar graph by densitometric analysis.
Figure 7.
CaM interacts with RV VP6 protein.
A. Predicted CaM-binding motif was found on RV-VP6 protein with high probability score in Calmodulin target Database. Putative CaM binding motif which spans from 271 to 292 was identified as NTYQARFGTIVARNFDTIRLSF. B & C. Position of putative CaM binding site in monomer (B) and trimer (C) structure of VP6 protein. D & E. Protein extract of HT29 cells at 0 hpi, 3 hpi and 9 hpi were incubated with appropriate bead conjugated antibody for overnight followed by detection with another antibody. Data shown here are representatives of three independent experiments. CaM antibody was used to pull down the complex followed by immunoblotting with VP6 (D). VP6 antibody was used to immunoprecipitate the complex followed by western blotting with CaM (E). F. pCDNA-VP6 and pCDNA-NSP3 were transfected individually and together in 293 cells followed by Co-IP with CaM antibody and immunoblotting with His antibody. CaM co-immunoprecipitated with VP6 but not with NSP3. G. Expression level of transfected pCDNA-VP6 and pCDNA-NSP3 in 293 cells were assessed by immunoblotting. Data shown in all the segments are representatives of three independent experiments.
Figure 8.
CaM-VP6 interaction is direct and Ca2+ dependent. A & B.
Affinity purified VP6 and purified CaM were incubated with appropriate bead conjugated antibody for overnight in a CaCl2 containing buffer with or without EGTA followed by detection with another antibody. Data shown here are representatives of three independent experiments. CaM antibody was used to pull down the complex followed by immunoblotting with VP6 (A). VP6 antibody was used to immunoprecipitate the complex followed by western blotting with CaM (B). C. Protein extract from HT29 cells at 0 hpi and 3 hpi were incubated overnight with bead conjugated anti-CaM antibody in a Co-IP buffer with or without BAPTA-AM followed by detection with anti-VP6 antibody. CaM level in immunoprecipitant were also shown. Data shown here are representatives of three independent experiments. D. Level of VP6 in BAPTA-AM treated and DMSO treated samples at 3 hpi of SA11 infection was analyzed. E. HT29 cells were either treated with BAPTA-AM or left untreated and infected for 3 hpi, 9 hpi, 12 hpi, 18 hpi and 24 hpi. Plaque assay were done for all samples, averaged results were expressed as (log (pfu/ml)). Data shown in all the segments are representatives of three independent experiments.
Figure 9.
Effect of W-7 on CaM-VP6 association and RV-SA11 titer.
A. HT29 cells either at 0 hpi or at 3 hpi were immunoprecipitated with CaM antibody. At 3 hpi cells were treated with BAPTA-AM, W-7 and DMSO separately. The immunoprecipitates were probed with VP6 antibody. Another one is left untreated at 0 hpi. B. HT29 cells treated with BAPTA-AM, W-7 and DMSO separately were lysed at 3 hpi. Another one is left untreated at 0 hpi. All samples were equally loaded in PAGE and immunoblotted with VP6 antibody. C. 0 hpi, 3 hpi and 9 hpi HT29 cells were either treated with W-7 or left untreated. Cells were lysed and immunoblotted with NSP3. D. HT29 cells were either treated with W-7 or left untreated and infected for 3 hpi, 9 hpi, 12 hpi, 18 hpi and 24 hpi. Plaque assay were done for all samples, averaged results were expressed as (log (pfu/ml)). Data shown in all the segments are representatives of three independent experiments.