Figure S2. A Translational SID-5::GFP Fusion Is Ubiquitously Expressed and Acts as a Dominant Negative. SID-5 Does Not Colocalize Extensively with GFP Markers of Golgi, Autophagosomes or Recycling Endosomes SID-5::GFP expression is detected in head neurons (A), excretory canal (B, H), intestine (C, D, J), spermatheca and somatic gonad (E, J), vulva (F), rectum (G), and canal associated neuron (I). All images show translational SID-5::GFP fusion except (D) which shows a transcriptional
sid-5p::GFP fusion with a nuclear localization signal. Nuclei are indicated by arrows in (C), (D) and (J). Bright spots are autofluorescent granules in the intestine, examples are indicated by arrowheads in (D), (H) and (J). (A-G) Left, fluorescence image; right, brightfield image. (H-I) fluorescence images. (J) The translational SID-5::GFP appears to localize to membranes in the spermatheca (Sp) and intestine. Top, fluorescence image; bottom, brightfield image. (K) Translational but not transcriptional GFP fusion of
sid-5 increases resistance to soaking RNAi of germline target
pal-1 in both wild-type and
sid-5(
qt24) background strains. Bars represent percent dead progeny laid 0-22 hours after soaking parents in 5 ug/ul
pal-1 dsRNA. n = number of treated parents.
sid-5p::GFP designates transcriptional fusion and
sid-5p::SID-5::GFP translational fusion. For
sid-5p::SID-5::GFP, the results for two independent lines are shown. Error bars represent SEM. (L) Immunohistochemistry using affinity-purified polyclonal rabbit SID-5 antibody (#2 in Fig. 2A) of intestines from wild-type animals (left) and strains carrying alleles
sid-5(
qt23) (center) or
sid-5(
qt34) (right). Top, immunofluorescence image; bottom, overlay with DAPI (blue), SID-5 channel pseudocolored in red. Scale bar 10 um.