Figure 4.

D76N β₂-m expression is prevented by RNA-interference (RNAi). Equal amounts of proteins (40 µg) were loaded on each lane and immunoblotted with polyclonal anti-human β₂-m antibody (a) and with anti-GAPDH (b). Lanes: M = Precision Plus Western C (Bio Rad) molecular weight standard. smg-1 incubated at 25 °C and collected at day 1 of adulthood. D76N β₂-m expressing nematodes incubated at 25 °C and collected at day 1 of adulthood and fed with HT115 bacteria transformed, as reported in the Methods section, with PAV2 plasmid for RNA-interfering (black box) or with control bacteria. Uncropped scans of immunoblots are shown in Supplementary Fig. S3. (c) Percentage of β₂-m expression is given as D76N β₂-m/GAPDH ratio of the WB band density of the RNAi strain relative to the control at their first day of adulthood. Density of the bands was determined by Image Studio Lite (LI-COR Biosciences). Three independent WB experiments were carried out and the results were plotted using GraphPad Prism (v6), p = 0.0052 vs the control level of expression (black) according to one sample t-test. (d) Index movement analysis. Three L4 worms were placed into NGM plates, fed with HT115/L4440 or HT115 bacteria transformed with PAV2 for RNA interference. Plates were maintained at 25 °C for 5 days. At this stage, the plates with adult worms and progenies were analysed by INVAPP Paragon software. Three independent experiments were carried out and the results were plotted using GraphPad Prism (v6). Data are mean of movement index parameter ± SEM; **p < 0.01 vs ctrl worms and °p < 0.05 vs the non-silenced D76N β₂-m expressing according to one-way Anova.