Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation

doi: 10.1016/j.xpro.2024.103297

Figure Lengend Snippet: Reagents and small molecules used in the β-like cell differentiation

Article Snippet: Sobetirome (GC-1) , Tocris , Cat# 4554.

Techniques: Concentration Assay, Solvent, Recombinant

Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation

doi: 10.1016/j.xpro.2024.103297

Figure Lengend Snippet:

Article Snippet: Sobetirome (GC-1) , Tocris , Cat# 4554.

Techniques: Recombinant, Membrane, Knock-Out, Electroporation, Passaging, Transfection, Control, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Pore Size

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 1. The promotion of spermatogonia activity by melatonin treatment. (A) Effects of melatonin treatment on the viability of GC-1 spg cells. The viability of spermatogonia cells were assessed by the CCK-8 method. (B, C) The enhancement of spermatogonia proliferation rates by treatments with melatonin. The Edu staining method was performed to measure GC-1 spg cell proliferation. (D, E) Inhibition of spermatogonia apoptosis by the treatments with melatonin. The per centages of GC-1 spg cells were quantitated by the flow cytometry. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Activity Assay, CCK-8 Assay, Staining, Inhibition, Flow Cytometry

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 2. Increased m6A RNA methylation levels in spermatogonia by melatonin. (A) Alterations of total m6A RNA methylation levels in GC-1 spg cells treated with 0.01, 0.1 or 1 μM mela tonin. The m6A contents in spermatogonia were quantitated by the RNA dot blot method. (B) Relative expression of major RNA methyl transferases and demethylases in GC-1 spg cells after melatonin treatments. Quantitative RT- PCR was done to assess target gene mRNA levels. (C) Significant increases of KIAA1429 protein levels in GC-1 spg cells induced by 0.01, 0.1 or 1 μM melatonin. KIAA1429 protein con tents in spermatogonia were determined via the western blot assay. GAPDH was detected as the internal standard. METTL3/4: methyltrans ferase-like protein 3/4; WTAP: Wilms’ tumor- associated protein; FTO: Fat mass and obesity- associated protein; ALKBH5: AlkB homolog 5. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Methylation, Dot Blot, Expressing, Quantitative RT-PCR, Western Blot, Wilms Tumor Assay

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 3. KIAA1429 mediates melatonin-induced spermatogonia activity changes. (A) Relative expression of KIAA1429 mRNA in spermatogonia with silenced or overexpressed KIAA1429 gene after melatonin treatment. KIAA1429 mRNA in GC-1 spg cells was detected by the quantitative RT-PCR. (B) Alterations of KIAA1429 protein levels in spermatogonia induced by KIAA1429 silencing or overexpression combined with melatonin treatment. Western blot was used to analyze KIAA1429 protein levels. (C) The changes of GC-1 spg cell viability induced by the combination of melatonin with KIAA1429 silencing or overexpression. Spermatogonia viability was assessed via CCK-8 assay. (D) Alterations of GC-1 spg cell proliferation rates caused by melatonin treatment together with KIAA1429 silencing or overexpression. Edu staining was done to analyze spermatogonia proliferation. (E) Effects of KIAA1429 silencing or overexpression on the apoptosis of GC-1 spg cells under melatonin treatment. Cell apoptosis was evaluated through the flow cytometry. si: silencing; ox: overexpression; ***P < 0.001; ###P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Over Expression, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 4. Transcriptome alterations in spermatogonia induced by KIAA1429 overexpression. (A) The scatter plots representing the differentially expressed genes in GC- 1 spg cells caused by KIAA1429 overexpression. Up-regulated and down-regulated genes in spermatogonia were showed as red and green spots respectively. (B) The functional categorization of differentially expressed genes in KIAA1429-overexpressing GC-1 spg cells. Differential gene categorization was done based on GO biological processes, cellular components and molecular functions. (C) The categorization of differentially expressed genes in KIAA1429-overexpressing GC-1 spg cells based on KEGG signaling pathways.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Over Expression, Functional Assay, Protein-Protein interactions

Journal: Reproductive biology

Article Title: Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m 6 A deposition to activate the PI3K/AKT signaling.

doi: 10.1016/j.repbio.2022.100681

Figure Lengend Snippet: Fig. 5. The roles of KIAA1429-induced PI3K/AKT activation in the regulation of spermatogonia activity by melatonin treatment. (A) Relative abundances of PI3K/ AKT signaling proteins in KIAA1429-overexpressing GC-1 spg cells without melatonin treatments. Signaling protein levels in spermatogonia were detected by western blot using GAPDH as the internal standard. (B) Relative abundances of PI3K/AKT signaling proteins in KIAA1429-overexpressing GC-1 spg cells under melatonin and LY294002 treatments. Signaling protein levels in spermatogonia were detected by western blot using GAPDH as the internal standard. (C) Effects of LY294002 on the viability of KIAA1429-overexpressing GC-1 spg cells under melatonin treatment. CCK-8 assay was used to assess spermatogonia viability. (D) Influences of LY294002 treatment on the proliferation rates of KIAA1429-overexpressing GC-1 spg cells treated with melatonin. Spermatogonia proliferation was quantitated by the Edu staining method. (E) Alterations of KIAA1429-overexpressing GC-1 spg cell apoptosis caused by LY294002 under melatonin treatment. PI3K: Phosphatidylinositol 3–kinase; AKT: Protein kinase B; *P < 0.05; **P < 0.01; ***P < 0.001; ###P < 0.001.

Article Snippet: To inhibit the PI3K signaling, GC-1 spg cells were treated with the 10 μM LY294002 (#HY-10108, MedChemExpress) which was added in the FBS-containing RPMI-1640 following the same procedure as above.

Techniques: Activation Assay, Activity Assay, Western Blot, CCK-8 Assay, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: OLFM4 expression is enhanced by Notch activation. Cells were stimulated with dH 2 O (control) or doxycycline (DOX, 100 ng/ml) for 24 h unless otherwise stated. (A) LS174T and DLD1 parent cells (Parent), and their respective tet-on NICD cells (NICD) were treated with DOX and collected for immunoblot analysis of NICD1 and Hes1. (B) Cells were treated with DOX and collected for qRT-PCR analysis of OLFM4 expression. data were normalized to β-actin levels. ** P < 0.01; **** P < 0.0001. n.s. not significant. (C) LS174T tet-on NICD cells were treated with DOX and collected for immunoblot analysis of intracellular OLFM4 protein. Two different forms of OLFM4 protein (B1 and B2) were observed.

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Activation Assay, Control, Western Blot, Quantitative RT-PCR

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: OLFM4 expression is enhanced by the synergy of TNF-α and Notch activation. (A) Cells were treated with DOX, TNF-α (50 ng/ml), IL-1β (25 ng/ml), IFN-γ (50 ng/ml), IL-6 (50 ng/ml), or LPS (100 ng/ml) for 24 h and collected for qRT-PCR analysis of OLFM4 expression. (B) After pre-treatment with DOX (100 ng/ml) for 24 h, LS174T cells were treated with TNF-α (50 ng/ml) for the indicated time-period. (C) After pre-treatment by DOX (100 ng/ml) for 24 h, LS174T cells were treated with TNF-α at the indicated concentration for 24 h. (D) Colonic organoids established from non-inflamed human colonic tissue (3 cases) were treated with TNF-α (50 ng/ml) for 24 h and collected for qRT-PCR analysis of OLFM4 expression. Data were normalized to β-actin levels. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Concentration Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: Increased expression of OLFM4 is regulated at the transcriptional level by TNF-α and Notch activation in human IECs.Cells were stimulated with dH 2 O (control) or DOX (100 ng/mL) for 24 h, unless otherwise indicated. (A) Luciferase reporter analysis using OLFM4-Luc. The transcriptional activity of the human OLFM4 gene was quantified in LS174T tet-on NICD and DLD1 tet-on NICD cells using a luciferase reporter plasmid containing the -2000 to +10 region of the human OLFM4 gene. (B) Luciferase reporter analysis using OLFM4-Luc with the addition of cytokines to LS174T tet-on NICD cells. (C) A ChIP assay for the human OLFM4 promoter region was performed in LS174T tet-on NICD cells. Cells were stimulated with DOX and TNF-α (50 ng/mL) for 24 h and subjected to ChIP analysis. Immunoprecipitation was performed using either rabbit IgG or anti-NICD1 antibodies. Primer sets were designed to amplify the proximal region of the human OLFM4 promoter, including an area with putative binding sites for RBP-Jκ and NF-κB (Site A). Data were normalized to the initial chromatin input. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, n.s. not significant.

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Activation Assay, Control, Luciferase, Activity Assay, Plasmid Preparation, Immunoprecipitation, Binding Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: Synergy between TNF-α and Notch activation promotes cytoplasmic accumulation of OLFM4 protein in human IECs. (A) LS174T tet-on NICD cells were treated with DOX and TNF-α (50 ng/ml) for 24 h, before the supernatants were collected for ELISA. The secretion levels of the OLFM4 protein are indicated. (B) LS174T tet-on NICD Cells were treated with DOX and TNF-α (50 ng/ml), IL-1β (25 ng/ml), or IFN-γ (50 ng/ml) for 24 h before immunoblot analysis. Protein levels of OLFM4, NICD1, and Hes1 are shown. (C) LS174T tet-on NICD Cells were treated with DOX and TNF-α (50 ng/ml) for 24 h before immunostaining for OLFM4 (green). Scale bar, 10 μm. (D) Apoptotic response under transient overexpression of cytoplasmic OLFM4 in LS174T cells. Cells were treated with TNF-α (50 ng/ml) for 24 h before collection for immunoblot analysis. Levels of PARP and OLFM4 are shown. * P < 0.05; n.s. not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining, Over Expression

Journal: Biochemistry and Biophysics Reports

Article Title: Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients

doi: 10.1016/j.bbrep.2020.100906

Figure Lengend Snippet: OLFM4 expression is enhanced and accumulates intracellularly in IECs of IBD patients. (A) Colonic organoids from patients were treated with TNF-α (50 ng/ml) for 24 h, before immunostaining for OLFM4 (green). (B) Inflamed and non-inflamed tissues from the small intestine and colon of patients were immunostained for OLFM4 expression (green). Small intestinal tissue of a patient with Crohn's disease and colon tissue of a patient with ulcerative colitis (UC) were used to show representative inflammatory patterns of OLFM4 expression. An enlarged view of an area of the left-side panel (white dotted square) is shown in the right panel. (C) Non-inflamed and inflamed colon tissues of a patient with UC were stained for OLFM4 (green), p65 (red), and NICD1 (red) using serial sections. Scale bar, 100 μm. All tissues or organoids were counterstained using DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The expression vector for OLFM4 was purchased from ORIGENE (RC214942).

Techniques: Expressing, Immunostaining, Staining

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 1. (a) Photo of the implantable medical grade polyether ether ketone (PEEK) capsules with a 250 μl drug reservoir and nanochan- nel membranes loaded with GC-1. (b) Scanning electron micro- scopic image of the nanofluidic membrane.

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques: Capsules, Membrane

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 2. Body weight (a), lean mass (b) and fat mass (c) (n = 10) for NMD–PBS- and NMD–GC-1-treated DIO mice (*Po0.05, **Po0.01 and ***Po0.001).

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques:

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 4. (a) TSH and (b) total T4 for (n = 10) NMD–PBS- and NMD–GC-1-treated DIO mice (***Po0.001).

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques:

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 3. Cholesterol (a), triglycerides (b), insulin (c) and glucose (n = 10) (d) for NMD–PBS- and NMD–GC-1-treated DIO mice (*Po0.05, **Po0.01 and ***Po0.001).

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques:

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 5. Thermogenic gene expression (n = 4) from white adipose tissue collected from (a) inguinal deposits (Wing), (b) WAT deposits near the NMD implant (Wimp) and from (c) BAT for NMD–GC-1-treated DIO mice normalized to PBS treatment (*Po0.10 and **Po0.05). Histology (hematoxylin–eosin (H&E)) for (d, g) WATing, (e, h) WATimp and (f, i) BAT for NMD–PBS- (d–f) and NMD–GC-1- (g–i) treated DIO mice. All images taken at original magnification × 10; scale bar is 100 μm.

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques: Gene Expression

Journal: International journal of obesity (2005)

Article Title: Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

doi: 10.1038/ijo.2016.129

Figure Lengend Snippet: Figure 6. Representative images of pancreatic islets from DIO mice treated with (a–c) NMD–PBS and (d–f) NMD–GC-1. Islets were double stained for insulin (green) and glucagon (red). All images taken at original magnification × 4; scale bar is 200 μm. Histomorphometric comparisons of (g) islet number and (h) β-cell area for DIO mice treated with NMD–PBS and NMD-GC-1 (*Po0.10). Four mice per group were analyzed.

Article Snippet: GC-1 (sobetirome) was purchased from Tocris (Bristol, UK).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors

doi: 10.1074/jbc.RA119.009712

Figure Lengend Snippet: Localization of outer segment-resident proteins in Cep290fl/fl;Cre+ mice. Localization of RHO (A), PRPH2 (B), ROM1 (C), ABCA4 (D), PDE6B (E), GUCY2D (F), ATP8A2 (G), and CNGA1 (H) was probed in Cep290+/fl;Cre+ (at P20) and Cep290fl/fl;Cre+ (at P20 and P40) retinas. I, retinal sections of Ift88+/fl;Cre+ (at P22) and Ift88fl/fl;Cre+ (at P22 and P40) mice were immunostained with anti-RHO antibody and shown for comparisons with Cep290fl/fl;Cre+ mice. Red brackets indicate outer segments. Sections were counterstained with DAPI to show nuclei (blue). At least three animals, both male and female, were used per group, and representative images are shown. Scale bar represents 50 μm. J, length of the outer segment in Cep290fl/fl;Cre+ and Ift88fl/fl;Cre+ retinas. Lengths of the outer segment were measured in areas 0.5–1.0 mm away from the optic nerve. Data are from five animals per genotype (two sections per animal). Mean and standard deviation (S.D.; error bar) are shown. Asterisk indicates statistical significance (two-tailed, two-sample t test assuming unequal variances; p < 0.01).

Article Snippet: Antibodies The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( 68 )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).

Techniques: Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors

doi: 10.1074/jbc.RA119.009712

Figure Lengend Snippet: Localization of inner segment membrane proteins in Cep290fl/fl;Cre+ mice. Retinal sections from Cep290+/fl;Cre+ (at P20) and Cep290fl/fl;Cre+ (at P20 and P40) mice were labeled with IMPG2 (A), HCN1 (B), and ATP1A3 (C) antibodies (green). Outer segments were delineated with ABCA4 or RHO (A), ATP8A2 (B), and GUCY2D (C) antibodies (red). Nuclei were counterstained with DAPI. D and E, localization of endomembrane proteins SYP (D) and LAMP1 (E) was examined using respective antibodies. At least three animals were used per group, and representative images are shown. Scale bar represents 50 μm. F, quantification of inner segment membrane protein mislocalization to the outer segment. Others are the same as in Fig. 4.

Article Snippet: Antibodies The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( 68 )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).

Techniques: Membrane, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors

doi: 10.1074/jbc.RA119.009712

Figure Lengend Snippet: Disruption of compartmentalized protein localization in Cep290rd16/rd16 mice. Localization of outer segment-resident proteins (PRPH2 (A), RHO (B and D), ROM1 (C), ABCA4 (E), GUCY2D (F), ATP8A2 (G), PDE6B (H), and CNGA1 (H); all in red except CNGA1, which is in green) and inner segment membrane proteins (STX3 (A), STXBP1 (B), SNAP25 (C), IMPG2 (D), VAMP2 (E), ATP1A3 (F), and HCN1 (G); all in green) was examined by indirect immunofluorescence microscopy. Scale bar represents 25 μm.

Article Snippet: Antibodies The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( 68 )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).

Techniques: Disruption, Membrane, Immunofluorescence, Microscopy