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. 2017 Dec 1;130(23):3975–3987. doi: 10.1242/jcs.205492

Fig. 1.

Fig. 1.

The Arf GEF GBF1 is localized at the trans-Golgi and interacts with Arf4, the Arf GAP ASAP1 and the ciliary cargo, rhodopsin. (A–D) Retinas were labeled with rabbit anti-GBF1 and mouse anti-GM130, followed by Cy3- and Cy5-conjugated secondary antibodies, followed by rabbit anti-Rab6 conjugated to Alexa Fluor 488. An individual optical section is shown. G, Golgi; N, nucleus. Scale bar: 3 µm. GBF1 (red) overlaps with the trans-Golgi marker Rab6 (green) (arrows), significantly better than with the cis-Golgi marker GM130 (blue), as per pixel colocalization analysis performed within the Golgi and expressed as the Pearson's coefficient (***P=3.42×10−5) (n=5 cells). (E) Schematic of a photoreceptor cell. The ROS is an elaborate primary cilium. Golgi and the TGN are localized in the myoid region (M) of the RIS. RTCs bud from the TGN and travel to the cilium (arrow), through the ellipsoid region (E) packed with mitochondria. Adherens junctions (AJ) form the outer limiting membrane (OLM) throughout the retina. (F) GBF1+Rab6 interaction sites (red dots) detected by PLA using mouse (m) anti-GBF1 and rabbit (r) anti-Rab6. Following the detection of interaction sites by PLA (arrows), sections were subsequently stained with an antibody against Rab6 conjugated to Alexa Fluor 488 (green). Nuclei were stained with TO-PRO-3 (blue). (G,H) PLA for GM130(m)+GBF1(r) (G) and P115(m)+GBF1(r) (H). (I) Golgi staining with Rab6(Alexa Fluor 488), p115(r) and GM130(m). (J–M) PLA for GBF1(m)+Arf4(r) (J), rhodopsin(m)+GBF1(r) (K), rhodopsin(m)+Arf4(r) (L) and ASAP1(m)+GBF1(r) (M). Scale bar: 5 µm. (N) Red dots were counted for the PLA pairs shown in panels J–M (30 cells each) in three separate experiments. The data from a representative experiment were expressed as a percentage of total interaction sites within the RIS, analyzed using Student's t-test (n=30) and presented as mean±s.e.m. (O) PNS (0.1 retina), or T/G/E, RTC and cytosolic fractions (0.25 retina each), were analyzed by immunoblotting (IB), as indicated. All antibodies were tested on a single blot.