Journal: The Journal of Biological Chemistry
Article Title: Targeted delivery of rhodopsin’s assembled core is required for outer segment extension in mouse rod photoreceptors
doi: 10.1016/j.jbc.2025.111106
Figure Lengend Snippet: Rhodopsin C-terminal truncations reveal unique localization profiles in WT mouse rods. A , the following FLAG-tagged rhodopsin constructs were electroporated into WT mouse rods: rhodopsin lacking its final five amino acids (RhoΔ5), 25 amino acids (RhoΔ25), or 38 amino acids (RhoΔ38). Δ5 n = 9, 35 images; Δ25 n = 10, 33 images; and Δ38 n = 6, 27 images. B , FLAG-tagged rhodopsin constructs immunoprecipitated from electroporated RhoKO mouse retinal lysates were treated with PNGase F or endo H and analyzed by Western blot. Untreated lysates (−), PNGase F (P), endo H (E). Resistance to endo H treatment indicates endoplasmic reticulum (ER) exit and normal processing through the conventional secretory pathway for FLAG-Rho, FLAG-RhoΔ5, and FLAG-RhoΔ25. Sensitivity to both PNGase F and endo H treatments shows increased electrophoretic mobility for FLAG-RhoΔ38, indicative of retention within the ER. Due to the reduced molecular weight of FLAG-RhoΔ38, the rhodopsin dimer (∼48 kDa) that persists under denaturing conditions was used to track the deglycosylation status of FLAG-RhoΔ38. Yellow asterisks indicate nonspecific bands. C , WT mouse rods were electroporated with FLAG-tagged, single-pass transmembrane domain fused to intact C-terminal cytosolic tail of rhodopsin (TM-RhoCT), or rhodopsin’s C-terminal tail lacking the final five amino acids (TM-RhoCTΔ5) or the final 25 amino acids (TM-RhoCTΔ25). TM-RhoCT n = 4, 41 images; TM-RhoCTΔ5 n = 6, 24 images; and TM-RhoCTΔ25 n = 5, 36 images. Bar graphs show the quotient between the OS signal over the total signal for each construct and are plotted next to the untargeted eGFP-TM from . FLAG staining ( green ), mCherry ( red ) labels transfected rod cells, and DAPI ( blue ) was used to counterstain nuclei. The scale bar represents 10 μm. DAPI, 4′,6-diamidino-2-phenylindole; eGFP, enhanced GFP; endo H, endoglycosidase H; IS, inner segment; N, outer nuclear layer; OS, outer segment; PNGase F, peptide- N -glycosidase F; RhoKO, rhodopsin KO; S, synapses; TM, transmembrane.
Article Snippet: For PNGase F treatment, the lysate is combined with 2 μl each glycoprotein denaturing buffer, GlycoBuffer 2, 10% NP-40, 1 μl PNGase F (P0708L, New England Biolabs), and H 2 O as necessary to a final volume of 20 μl.
Techniques: Construct, Immunoprecipitation, Western Blot, Molecular Weight, Staining, Transfection
Genovis Inc is a verified supplier 
Genovis Inc manufactures this product 
