Fig 1: RIC-3 knockdown in SH-SY5Y cells.A For control experiments, SH-SY5Y cells treated with 5 nM Silencer GAPDH siRNA (ThermoFisher Scientific, Cat# 4390849), achieved approximately 95% GAPDH knockdown (KD). The cells were incubated for 72 hours at a confluence level of 50%. B RIC-3 protein knockdown was performed by incubating SH-SY5Y cells at 50% confluence with 5 nM RIC-3 siRNA for 24 hours. The first Western blot (left) shows the control (RIC-3; 50 kDa, lane 9) and siRNA-treated samples for RIC-3 (RIC-3 KD; lane 10). To validate the knockdown, we used calnexin (100 kDa; lane 9–10) as an internal loading control by probing the blot shown on the left with calnexin antibody to yield the blot on the right. C-D RIC-3 knockdown reduces plasma membrane surface expression of nACh α7 and 5HT3A receptors in SH-SY5Y cells. Western blot analysis was performed on plasma membrane (PM) fractions isolated from SH-SY5Y cells either transfected with RIC-3 siRNA (RIC-3 KD) or untransfected (RIC-3). Blots were probed with antibodies against C 5-HT3A (50 kDa) and D nAChRα7 (50kDa) subunits. Both showed approximately 98% reduction in the PM fraction of RIC-3 knockdown cells compared to control. Na+/K+-ATPase (100 kDa) was used as a reference protein for sample loading normalization. All blots were quantified and normalized using Bio-Rad ImageLab software. Blots in Fig. 3 were cropped for clarity and presentation purposes. Molecular-weight marker positions (in kDa) are indicated on the sides of each blot.
Fig 2: Identification of RIC-3 following affinity enrichment using immobilized L1-MX peptide.A Pull-down assay procedure. I L1-MX peptide containing an N- or C-terminal Cysteine (‘bait’) and the iodoacetyl resin react to form a covalent bond yielding L1-MX peptide alkylated with resin as shown in II during the coupling process. III Tissue or cell extract containing RIC-3 protein are incubated with the modified resin and RIC-3 will bind to the linked L1-MX peptide. IV Elution with SDS denatures this complex to release bound proteins/RIC-3 that had interacted with L1-MX peptide. V Eluates are separated by denaturing SDS-PAGE and RIC-3 detected using immunoblotting. B-E Pull-down results for B Xenopus laevis oocytes PM, C mouse brain ER, D SHSY5Y ER and cytosol (C) fractions and E ER fractions from transfected SH-SY5Y cells treated with 5 nM RIC-3 siRNA. Pull-downs from oocytes B and mouse brain C confirmed RIC-3 binding to L1MX (50 kDa; lane 4 and 7, respectively). In SHSY5Y cells, cytosol lysates D (lane 7–8) did not recover RIC-3, indicating that enrichment was specific to the ER fraction. ER-derived RIC-3 (100 kDa; lane 5–6) bound ~3-fold more strongly to L1-MX than to the Cys-resin control D. In panel E, RIC-3 interacts with L1-MX in non-transfected cells (100 kDa band, lane 9), but not in RIC-3 knockdown samples (lane 8). Faint additional bands at ~50 and ~75 kDa (lanes 4, 6–9) represent nonspecific signals. Blots in panels B-E were cropped for clarity and presentation purposes. Molecular-weight marker positions (kDa) are indicated on the sides of each blot.
Fig 3: Subcellular fractionation of mouse brain tissue or SH-SY5Y cells.Illustration of the fractionation procedure for isolating crude plasma membrane (PM) and endoplasmic reticulum (ER) fractions from A mouse brain or B SH-SY5Y neuroblastoma cells. Additionally, cytosolic (C), and crude mitochondrial (M) fractions are obtained. Red star denotes samples used for subsequent analyses. C1-C6 depicts differential centrifugations. C Subcellular localization of RIC-3 in mouse brain and SH-SY5Y cells; validation of fraction purity. RIC-3 protein was probed in cytosol (C), endoplasmic reticulum (ER), mitochondria (M), and crude plasma membrane (PM) fractions using a mouse anti-RIC-3 primary antibody (Abnova, cat# H00079608-B01P) and an HRP-conjugated goat-raised anti-mouse secondary antibody (Thermo Fisher Scientific, Cat. #31430). Analyses were performed using fractionated mouse brain tissue C and SH-SY5Y cells D. Human recombinant RIC-3 (hRIC-3) and RIC-3 (both ~50 kDa) expressed in Xenopus laevis oocytes were included as positive controls. In both systems, the strongest RIC-3 (~50 kDa) signal was observed in the ER fraction. In mouse ER fractions (panel C), RIC-3 appears as a doublet at ~50 kDa (lane 6), consistent with post-translational modification. Fraction purity was validated using calnexin (ER; Invitrogen cat# PA5–34754; ~100 kDa), Na+/K+-ATPase (PM; Cell Signaling Technologies cat# 3010S; ~100 kDa) and GADPH (C; Cell Signaling Technologies, 5174; ~35 kDa). HRP-conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific, cat# 31460) was used for rabbit-raised primaries. Blots shown in panels C and D (to the right of the arrow) were performed sequentially on separate days. Blots are semiquantitative, and ER signal bleed-through in non-ER fractions was corrected using a Calnexin-based linear contamination model. Additional RIC-3 reactive bands in mouse brain/SH-SY5Y cells correspond in size to mouse and human RIC-3 isoforms in the Uniprot database.
Fig 4: Glycosylation of RIC-3.A A double band of RIC-3 (50 kDa; lane 2) is observed in the mouse ER fraction. This fraction was subjected to deglycosylation using PNGase F (F: RIC-3) and Endo H (H: RIC-3) under non-denaturing conditions. PNGase F treatment resulted in the disappearance of the upper RIC-3 band (lane 3), indicating the removal of N-linked glycans. Endo H had no effect on RIC-3 (lane 4), suggesting resistance to Endo H-sensitive glycan cleavage. B Comparison of mouse RIC-3 electrophoretic profiles in ER and PM fractions. In both the gradient 4–20% (blot on the right, lane 6) and 10% gels (blot on the left, lane 3), ER-localized RIC-3 appears as characteristic upper and lower bands (~50 kDa). In contrast, the PM fraction shows a prominent upper band with little to no lower band on the gradient gel (~50 kDa, lane 5). When resolved on a 10% gel, the PM fraction displays multiple faint bands (lane 4). The ~25 kDa bands present in both fractions likely represent a processed or fragmented form of RIC-3. Blots in Fig. 5 were cropped for clarity and presentation purposes. Molecular-weight marker positions (kDa) are indicated on the sides of each blot.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for RIC3 Recombinant Protein Antigen