rabbit anti rbp1 antibody  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: The role of RBP1 in the visual cycle and generation of Abca4 −/− /Rdh8 −/− /Rbp1 −/− mouse l ines. A , schematic representation of the visual cycle, illustrating the cellular and molecular components involved in visual chromophore regeneration. Proteins are color-coded based on their roles: enzymes ( blue ), transporters and receptors ( purple ), and retinoid carriers ( green ). Rhodopsin is shown in gray , pink , and yellow depending on its state, while RBP1 is highlighted in red . B , representative agarose gels showing genotyping results for Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice and their heterozygous and WT counterparts. C , western blot analysis of ocular tissues from Rbp1 −/− mice. Unlike in Abca4 −/− /Rdh8 −/− (DKO) controls, RBP1 is absent in eyeball homogenates of Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice. D , immunofluorescence-based confirmation of RBP1 deficiency. Retina sections were probed with an anti-RBP1 antibody, revealing specific staining exclusively in the RPE cell layer of Abca4 −/− /Rdh8 −/− mice. No immunofluorescence signal was detected in the corresponding Rbp1 −/− mice on the Abca4 −/− /Rdh8 −/− (DKO) genetic background. The scale bar represents 50 μm. E , the retinal morphology of 4-month-old mice showed no signs of spontaneous retinal degeneration in either mouse line. The scale bar represents 150 μm. PR (photoreceptors), ONL (outer nuclear layer), OPL (outer plexiform layer), INL (inner nuclear layer), IPL (inner plexiform layer); GCL (ganglion cell layer). F , quantification of 11c-RAL in the retinas of Abca4 −/− /Rdh8 −/− (DKO) and Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice. No statistically significant (ns) differences were observed in visual chromophore content, indicating comparable levels of rhodopsin (n = 6). G , the effect of RBP1 deficiency on the rate of dark adaptation. Scotopic ERG responses were recorded after 90 min of dark adaptation following exposure to a bright light flash that bleached ∼80% of the rod visual pigment. Delayed dark adaptation in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice was evidenced by reduced ERG a- and b-wave amplitudes compared to Abca4 −/− /Rdh8 −/− (DKO) mouse line. The absence of RDH8 and ABCA4 alone had a detectable, but much subtler effect on the regeneration of visual pigment, as observed for Abca4 +/+ /Rdh8 +/+ /Rbp1 +/+ mice, indicated as “WT”. Data represent mean ± S.D. (n = 6). Student’s t -test was used for two-group comparisons of individual experimental points (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005). 11c-RAL, 11-cis-retinal; ERG, electroretinogram; RBP1, cellular retinol-binding protein 1; RPE, retinal pigment epithelium.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Western Blot, Immunofluorescence, Staining, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Ocular phenotype of Rbp1 −/− mice on the Abca4 −/− /Rdh8 −/− genetic backgrounds. A , schematic representation of the experimental design. The light intensity and exposure duration were chosen to induce ∼80% reduction of the retinal ONL in Abca4 −/− /Rdh8 −/− mice. B , representative SLO images of retinas of 4-month-old mice after bright light exposure. Increased autofluorescence with a characteristic punctate pattern indicates immune cell infiltration into the damaged retina of Abca4 −/− /Rdh8 −/− (DKO) mice, a feature not observed in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice. C , quantification of the SLO signal shown in panel ( B ) reveals a statistically significant difference (∗∗∗ p < 0.0005, unpaired t test) between DKO and TKO mice (9 ≤ n ≤ 14). D , retinal integrity assessment in light-exposed mice using OCT. ONL thickness was measured to quantify the degree of retinal degeneration. The scale bar represents 50 μm. E , quantification of retinal morphology changes based on OCT images. The data indicate partial protection against light-induced damage in RBP1-deficient mice ( Abca4 −/− /Rdh8 −/− /Rbp1 −/− labeled as TKO) compared to their Abca4 −/− /Rdh8 −/− (DKO) control counterparts. The prebleach data for DKO and TKO mice are depicted in gray and white triangles, respectively. Data represent mean ± S.D. (12 ≤ n ≤ 16). Asterisks indicate statistical significance between individual points (∗∗∗ p < 0.0005, unpaired t - test). F , representative histology images corresponding to the OCT data shown in panel ( D ) confirm the protective effect of the Rbp1 gene deletion. G , ERG-based functional assessment of retinal responses after exposure to damaging bright light. While Abca4 −/− /Rdh8 −/− mice exhibited diminished light responses, retinal function was largely preserved in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t test for two-group comparisons at individual experimental points). The prebleach data represent combined ERG responses from both mouse lines. Data represent mean ± S.D. (n = 6). ERG, electroretinogram; OCT, optical coherence tomography; ONL, outer nuclear layer; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Labeling, Control, Functional Assay, Tomography, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Deletion of the Rbp1 gene decreases light sensitivity of Balb/cJ albino mice. A , schematic representation of the experimental design. Balb/cJ mice were exposed to bright light for 55 min, resulting in ∼50% reduction in ONL thickness. B , SLO images of retinas from 4-month-old albino mice after bright light exposure. Increased autofluorescence was readily observed in Rbp1 + / + (Balb/cJ) mice but was greatly diminished in Rbp1 −/− (Balb/cJ) mice, indicating a decreased in immune cell infiltration. C , quantification of the SLO signal revealed a statistically significant difference between Rbp1 +/+ (Balb/cJ) and Rbp1 −/− (Balb/cJ) mice (∗∗∗ p < 0.0005, unpaired t test, n = 6). D , quantification of ONL thickness in light-exposed mice. The absence of RBP1 in the light-sensitive albino mice led to partial preservation of retinal morphology (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t test for two-group comparisons for individual experimental points). The prebleach data represent combined ONL thickness from both Rbp1 +/+ (Balb/cJ) and Rbp1 −/− (Balb/cJ) mouse lines. The data represent mean ± SD (n = 10). E , preservation of retinal morphology in Rbp1 −/− mice, as shown in panel ( D ), correlates with sustained retinal function, particularly at higher light intensities, as assessed by scotopic ERG responses (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t - test for individual experimental points). Data represent mean ± S.D. (n = 6). ERG, electroretinogram; ONL, outer nuclear layer; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Preserving, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Temporal accumulation of A2E in mouse eyes. A , experimental design. Abca4 −/− /Rdh8 −/− (DKO), Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO), and corresponding WT mice were maintained under a 12 h/12 h light/dark cycle for up to 8 months to assess the rate of A2E accumulation. B , LC/MS-based detection and quantification of A2E. Synthetic A2E-d10 was used as an internal standard to determine endogenous A2E levels. Single reaction monitoring mode was employed for the unambiguous identification of endogenous and synthetic A2E molecules in eye extracts. C , time-dependent increase of A2E levels in DKO, TKO, and WT mouse eyes. The calculated rate of A2E accumulation was approximately three times slower in Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) mice compared to Abca4 −/− /Rdh8 −/− (DKO) controls. Data represent mean ± S.D. (4 ≤ n ≤ 7). D , Box-and-whisker plot illustrating the distribution of experimental data for samples collected from 4-month-old and 8-month-old mice, presented in panel ( C ) (∗∗∗ p < 0.0005, unpaired t - test for two-group comparisons). E , representative SLO images of 8-month-old mouse retinas, showing differences in autofluorescence. The increased SLO signal across the retina correlates with higher A2E accumulation in Abca4 −/− /Rdh8 −/− (DKO) mice compared to Abca4 −/− /Rdh8 −/− /Rbp1 −/− (TKO) animals. F , the quantification of the SLO signal is shown in panel ( E ) (n = 8, ∗∗∗ p < 0.0005, unpaired t test for two-group comparisons). A2E, N -retinylidene- N -retinylethanolamine; LC/MS, liquid chromatography/mass spectrometry; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Liquid Chromatography with Mass Spectroscopy, Whisker Assay, Liquid Chromatography, Mass Spectrometry, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: Inhibitors of RBP1 protect against acute light-induced retinal degeneration in Abca4 −/− /Rdh8 −/− mice. A , experimental design. RBP1 inhibitors were administered intraperitoneally to dark-adapted mice 30 min before exposure to retinal-damaging light. B , chemical structures of the inhibitors used in this study. Apparent K i values highlight the difference in potency between abn-CBD and oxadiazol-derived compounds. C , effect of light exposure on retinal integrity, measured by ONL thickness. Pretreatment with abn-CBD, a potent RBP1 inhibitor, provided robust protection against acute retinal degeneration (∗∗∗ p < 0.0005, unpaired t test for two-group comparisons at each experimental point). Data represent mean ± S.D. D , retinal autofluorescence after light exposure, assessed by SLO in abn-CBD-treated and untreated (control) mice. White puncta in the untreated group indicate inflammatory changes in the retina. E , quantification of SLO brightness from panel ( D ), showing a statistically significant difference between treated and untreated groups (∗ p < 0.05, unpaired t - test, n = 6). F , scotopic ERG recordings reveal statistically significant preservation of retinal function in mice pretreated with abn-CBD (∗ p < 0.05, unpaired t test, n = 6). abn-CBD demonstrated efficacy under high-intensity light exposure, which caused ∼80% photoreceptor degeneration in untreated mice. Data represent mean ± S.D. G , comparison of the protective effects of alternative RBP1 inhibitors. The efficacy of the less potent inhibitors (1–3, panel B ) was evident only under conditions of reduced light exposure (ONL thickness reduction of ∼30%). Under these conditions, post hoc analysis showed significant differences between untreated animals and those injected with RBP1 inhibitors. Among the alternatives, inhibitor two exhibited the most profound retinal protective effect, based on statistical analysis (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005, unpaired t test for individual experimental points). Data represent mean ± S.D. (n = 4). abn-CBD, abnormal cannabidiol; ERG, electroretinogram; ONL, outer nuclear layer; RBP1, cellular retinol-binding protein 1; SLO, scanning laser ophthalmoscopy.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Derivative Assay, Control, Preserving, Comparison, Injection, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inactivation of cellular retinol-binding protein 1 protects against bis-retinoid accumulation and light-induced retinal degeneration in mice

doi: 10.1016/j.jbc.2025.110538

Figure Lengend Snippet: The role of RBP1 in retinoid homeostasis and the mechanistic basis for the protective effect of its inhibitors. RBP1 facilitates the intracellular transport of at-ROL in RPE cells. Its absence delays the regeneration of the visual chromophore by slowing down at-ROL esterification by LRAT, leading to a transient accumulation of at-ROL compared to WT mice. An unforeseen consequence of RBP1 deficiency is diminished light sensitivity in mice susceptible to light-induced retinal damage, along with a slower accumulation of at-RAL-derived side metabolites. Pharmacological inhibition of RBP1 mimics the phenotype of Rbp1 −/− mice, offering a potential therapeutic strategy for modulating the visual cycle. Therefore, RBP1 inhibitors may provide an alternative approach to treating diseases associated with impaired clearance of at-RAL and excessive bis-retinoid accumulation. LRAT, lecithin:retinol acyltransferase; RBP1, cellular retinol-binding protein 1; RPE, retinal pigment epithelium.

Article Snippet: Up to 2 μg of total RNA was reverse transcribed into complementary DNA in a 20 μl reaction using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using TaqMan probes (Applied Biosystems) for Rbp1 (Mm00441119_m1), with Gapdh (Mm99999915_g1) serving as an endogenous control.

Techniques: Derivative Assay, Inhibition, Binding Assay

Journal: Neural Regeneration Research

Article Title: AAV2- PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis

doi: 10.4103/NRR.NRR-D-23-01301

Figure Lengend Snippet: Sequence of primers used in this study

Article Snippet: The antibodies were as follows: rabbit anti-PDE6B antibody (1:500, Thermo Fisher Scientific, Cat# PA1-722, RRID: AB_2161443), rabbit anti-PDE6A antibody (1:500, Thermo Fisher Scientific, Cat# PA1-720, RRID: AB_2268019), rabbit anti-Rom1 antibody (1:1000, Cusabio, Wuhan, China, Cat# CSB-PA344843ESR2HU, RRID: AB_3076310), rabbit anti-Efcbp2 antibody (1:1000, Novus Biologicals, CO, USA, Cat# Nbp1-84002, RRID: AB_11028373), rabbit anti-Aldh1a1 antibody (1:500, Huabio, Hangzhou, China, Cat# ET1605-24, RRID: AB_3069708), rabbit anti-Rhodopsin antibody (Rho; 1:1000, Huabio, Cat# ET1704-12, RRID: AB_3070464), rabbit anti-Sec63 antibody (1:500, Proteintech, Wuhan, China, Cat# 13978-1-AP, RRID: AB_2186546), rabbit anti-Rbp1 antibody (1:1000, Proteintech, Cat# 22683-1-AP, RRID: AB_11182381), mouse anti-Bcl2 antibody (1:1000, Proteintech, Cat# 60178-1-LG, RRID: AB_10734459), rabbit anti-Bax antibody (1:1000, Abcam, Cambridge, MA, USA, Cat# ab32503, RRID: AB_725631), rabbit anti-ERK1/2 antibody (1:1000, Cell Signaling Technology, Danvers, CO, USA, Cat# 4695, RRID: AB_390779), rabbit anti-phospho-ERK1/2 antibody (1:2000, Cell Signaling Technology, Cat# 4370, RRID: AB_2315112), rabbit anti-c-Fos antibody (phospho Ser32, 1:500, Immunoway, Plano, TX, USA, YP0442, RRID: AB_3076316), rabbit anti-c-Fos antibody (1:500, Huabio, Cat# ET1701-95, RRID: AB_3070261), rabbit anti-GAPDH antibody (1:1000, Proteintech, Cat# 10494-1-AP, RRID: AB_2263076), and rabbit anti-β-actin antibody (1:1000, Cell Signaling Technology, Cat# 4970, RRID: AB_2223172).

Techniques: Sequencing