Journal: Acta Histochemica et Cytochemica

Article Title: Correlation between the Appearance of Neuropeptides in the Rat Trigeminal Ganglion and Reinnervation of the Healing Root Socket after Tooth Extraction

doi: 10.1267/ahc.05057

Figure Lengend Snippet: SP-, NK 1 -R-, BDNF-, TrkB-, and ATF3-immunoreactive neurons in the trigeminal ganglion. ( a, c ) SP-, ( e, g ) NK 1 -R-, ( i, k ) BDNF-, ( m, o ) TrkB- and ( q ) ATF3-immunoreactive neurons in the maxillary nerve regions in the non-operated rat trigeminal ganglion ( a, e, i, m ) and at 3 days after extracted rat trigeminal ganglion ( c, g, k, o, q ). ( b, d, f, h, j, l, n, p, r ) Immunohistochemical images for PGP-9.5 in the same region as a, c, e, g, i, k, m, o, q . Bar=50 µm.

Article Snippet: Then, the sections were incubated with rabbit polyclonal antibodies against SP (1:1000; Affiniti Research Products, Devon, UK), rabbit polyclonal antibodies against NK 1 -R (1:1000; Calbiochem–Novabiochem, San Diego, CA, USA), sheep polyclonal ­antibodies against BDNF (1:100; Chemicon International, Temecula, CA, USA), rabbit polyclonal antibodies against TrkB (1:2000; Chemicon International) or rabbit polyclonal antibodies against ATF3 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hr at 37°C.

Techniques: Immunohistochemical staining

Journal: Acta Histochemica et Cytochemica

Article Title: Correlation between the Appearance of Neuropeptides in the Rat Trigeminal Ganglion and Reinnervation of the Healing Root Socket after Tooth Extraction

doi: 10.1267/ahc.05057

Figure Lengend Snippet: SP- and NK 1 -R immunoreactive neurons in the rat trigeminal ganglion after 3 days following extraction. ( a ) NK 1 -R- and ( b ) PGP-9.5-immunoreactive neurons in the maxillary nerve region. ( e ) SP- and ( f ) NK 1 -R-immunoreactive neurons in the maxillary nerve region. ( c, g ) Differential interference contrast image in the same region as a, b and e, f respectively. ( d, h ) are merged image of ( a, b, c ) and ( e, f, g ). The cells indicated by orange color represent immunopositive for both SP and NK 1 -R. Bar=100 µm.

Article Snippet: Then, the sections were incubated with rabbit polyclonal antibodies against SP (1:1000; Affiniti Research Products, Devon, UK), rabbit polyclonal antibodies against NK 1 -R (1:1000; Calbiochem–Novabiochem, San Diego, CA, USA), sheep polyclonal ­antibodies against BDNF (1:100; Chemicon International, Temecula, CA, USA), rabbit polyclonal antibodies against TrkB (1:2000; Chemicon International) or rabbit polyclonal antibodies against ATF3 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hr at 37°C.

Techniques:

Journal: Acta Histochemica et Cytochemica

Article Title: Correlation between the Appearance of Neuropeptides in the Rat Trigeminal Ganglion and Reinnervation of the Healing Root Socket after Tooth Extraction

doi: 10.1267/ahc.05057

Figure Lengend Snippet: The ratio of NK 1 -R- ( a ), BDNF- ( b ), and TrkB-immunoreactive neurons per PGP-9.5-immunoreactive neuron in the maxillary ( a, c, e ) and mandibular ( b, d, f ) nerve regions between 3 hr and 21 days after extraction. Cont., control; Ext, extracted groups; Max, maxillary nerve region; Md, mandibular nerve region. Data are expressed as the mean±SD. Control group n=4. Experimental group n=7. ** p<0.01, * p<0.05.

Article Snippet: Then, the sections were incubated with rabbit polyclonal antibodies against SP (1:1000; Affiniti Research Products, Devon, UK), rabbit polyclonal antibodies against NK 1 -R (1:1000; Calbiochem–Novabiochem, San Diego, CA, USA), sheep polyclonal ­antibodies against BDNF (1:100; Chemicon International, Temecula, CA, USA), rabbit polyclonal antibodies against TrkB (1:2000; Chemicon International) or rabbit polyclonal antibodies against ATF3 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hr at 37°C.

Techniques:

Journal: Acta Histochemica et Cytochemica

Article Title: Correlation between the Appearance of Neuropeptides in the Rat Trigeminal Ganglion and Reinnervation of the Healing Root Socket after Tooth Extraction

doi: 10.1267/ahc.05057

Figure Lengend Snippet: NK 1 -R immunoreactive osteoblasts (arrowheads) on the bone surface at the root socket. AB, alveolar bones; PDL, periodontal ligaments. Bar=20 µm.

Article Snippet: Then, the sections were incubated with rabbit polyclonal antibodies against SP (1:1000; Affiniti Research Products, Devon, UK), rabbit polyclonal antibodies against NK 1 -R (1:1000; Calbiochem–Novabiochem, San Diego, CA, USA), sheep polyclonal ­antibodies against BDNF (1:100; Chemicon International, Temecula, CA, USA), rabbit polyclonal antibodies against TrkB (1:2000; Chemicon International) or rabbit polyclonal antibodies against ATF3 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hr at 37°C.

Techniques:

Journal:

Article Title: Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity

doi: 10.1172/JCI200317808

Figure Lengend Snippet: P5 retinal whole-mount in situ hybridization of VEGF-A, VEGFR-1, and VEGFR-2 mRNA’s. (a) VEGF-A mRNA (pink) is detected anterior to the growing vessel front as outlined by (b) FITC-dextran perfusion of vessels (yellow green). Posterior to the vessel front, VEGF-A expression is suppressed (light yellow). (c) VEGFR-1 mRNA (pink) is detected in the central retina but is not seen at the anterior edge of vessels as outlined by (d) FITC-dextran perfusion of vessels (yellow). (e) VEGFR-2 mRNA (pink) is detected in the entire retina and does not correspond to the vessels (f) outlined in yellow.

Article Snippet: The sections were stained with primary antibodies against VEGFR-1 or VEGFR-2 (R&D Systems Inc.), and biotinylated Griffonia simplicifolia , thereafter G. simplicifolia , lectin I (endothelial cell specific) (Vector Laboratories Inc., Burlingame, California, USA) for 2 hours at room temperature.

Techniques: In Situ Hybridization, Expressing

Journal:

Article Title: Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity

doi: 10.1172/JCI200317808

Figure Lengend Snippet: Real-time RT-PCR quantification of VEGFR-1 and VEGFR-2 mRNA during retinal vascular development. Copy numbers of VEGFR-1 and VEGFR-2 mRNA/106 copies cyclophilin A control mRNA at specific timepoints were measured. (a) VEGFR-1 mRNA expression increases linearly with retinal vascular development; expression is 60-fold higher at P26 than at P3. (b) VEGFR-2 mRNA expression decreases modestly (<15%) during retinal vessel development. The ratio of VEGFR-2 mRNA to VEGFR-1 mRNA expression ranges from 200-fold at P3 to twofold at P26.

Article Snippet: The sections were stained with primary antibodies against VEGFR-1 or VEGFR-2 (R&D Systems Inc.), and biotinylated Griffonia simplicifolia , thereafter G. simplicifolia , lectin I (endothelial cell specific) (Vector Laboratories Inc., Burlingame, California, USA) for 2 hours at room temperature.

Techniques: Quantitative RT-PCR, Expressing

Journal:

Article Title: Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity

doi: 10.1172/JCI200317808

Figure Lengend Snippet: Immunohistochemical localization of VEGFR-1 and VEGFR-2 protein in P2 and P5 retinal whole mounts. On P5, VEGFR-1 protein (red) is detected in a vascular pattern (a and c) and clearly coincides with endothelial cells (blue) as detected with G. simplicifolia I isolectin (d). (e) A merged image (purple) shows coincidence. (b and f) VEGFR-2–positive signal (red) is found in the neural retina, specifically in the interstices between vessels (blue) (g) in the merged image (h), which shows little or no overlap between vessels and VEGFR-2 staining. On P2, VEGFR-1 protein signal (green) (i) coincides with endothelial cells (red) (j) detected by lectin. In a merged image (k), retinal vessels coincide with the faint VEGFR-1 staining. The retinal vessel front is defined by an arrow. Some hyaloid vasculature (H) from incomplete removal on the vitreal surface of the P2 retina is also positive for VEGFR-1 signal. (l) VEGFR-2–positive signal (green) is found in the neural retina and does not coincide with endothelial capillaries (red) (m) in the merged image (n). Arrows indicate the vessel front.

Article Snippet: The sections were stained with primary antibodies against VEGFR-1 or VEGFR-2 (R&D Systems Inc.), and biotinylated Griffonia simplicifolia , thereafter G. simplicifolia , lectin I (endothelial cell specific) (Vector Laboratories Inc., Burlingame, California, USA) for 2 hours at room temperature.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity

doi: 10.1172/JCI200317808

Figure Lengend Snippet: Immunohistochemical localization of VEGFR-1 and VEGFR-2 protein in P5 and P15 retinal cross sections. (a–f) P5 immunohistochemical localization of (a) VEGFR-1, (d) VEGFR-2, (b and e) G. simplicifolia I isolectin–stained endothelial cells, and (c and f) merged images. (a) VEGFR-1 protein (green) is seen primarily in the ganglion cell layer (GCL) and is seen to overlap with (b) endothelial cells (red) when (c) the images are merged (yellow; indicated by arrows). (d) VEGFR-2–positive signal (green) is found in the GCL, the inner plexiform layer, the inner nuclear layer (INL), and the outer nuclear layer (ONL). (e) Vascular endothelial cells (red) do not overlap with VEGFR-2–positive cells in the merged image (f) and are not coincident with vessels (arrows). (g–l) P15 immunohistochemical localization of (g) VEGFR-1 and (j) VEGFR-2 staining and (h and k) isolectin-stained endothelial cells. (i and l) Merged images. VEGFR-1 protein (green in g) completely overlaps with endothelial cells (red in h) when the images are merged (yellow in i). Some VEGFR-2 signal (green in j) overlaps with endothelial cells (k) when the images are merged (yellow in l), whereas other VEGFR-2–positive cells (indicated by arrow in d and f) span the retina and are morphologically consistent with Muller cell structure. RPE/Ch, retinal pigment epithelium/choroid.

Article Snippet: The sections were stained with primary antibodies against VEGFR-1 or VEGFR-2 (R&D Systems Inc.), and biotinylated Griffonia simplicifolia , thereafter G. simplicifolia , lectin I (endothelial cell specific) (Vector Laboratories Inc., Burlingame, California, USA) for 2 hours at room temperature.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity

doi: 10.1172/JCI200317808

Figure Lengend Snippet: PlGF-1, but not VEGF-E, prevents hyperoxia-induced retinal vessel loss, thus implicating VEGFR-1 in survival. PlGF-1: P8 FITC-dextran–perfused retinal flat-mount retina from a representative control mouse treated with room air (normoxia) (a) or a mouse given hyperoxic treatment (75% O2 for 17 hours at P7–P8) after intravitreal injection on P7 of (b) control BSS in one eye and (c) the VEGFR-1–specific ligand PlGF-1 in the contralateral eye. Vessels delineated with FITC show that PlGF-1 confers significant protection from oxygen-induced vessel loss compared with BBS control. (d) Analysis of nonvascularized area shows a greater than fourfold difference between eyes treated with PlGF-1 (22.2% ± 3.4% vascularized area) and eyes treated with BSS (5.1% ± 1.2%) (n = 6, P < 0.001). VEGF-E: FITC-dextran–perfused retinal flat mount of P8 control retina from representative room air–treated mouse (e) or oxygen-exposed mouse after intravitreal injections at P7 of (f) control BSS in one eye and (g) VEGFR-2–specific ligand VEGF-E in the contralateral eye. (h) Analysis of nonvascularized area shows no significant difference between VEGF-E– and BSS-treated eyes (n = 6, P = 0.87). Results are representative of two independent experiments.

Article Snippet: The sections were stained with primary antibodies against VEGFR-1 or VEGFR-2 (R&D Systems Inc.), and biotinylated Griffonia simplicifolia , thereafter G. simplicifolia , lectin I (endothelial cell specific) (Vector Laboratories Inc., Burlingame, California, USA) for 2 hours at room temperature.

Techniques: Injection

Journal:

Article Title: Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity

doi: 10.1172/JCI200317808

Figure Lengend Snippet: Activation of VEGFR-1 by PlGF-1 does not increase normal retinal vessel growth or revascularization. (a) Intravitreal injections at P3 of control (BSS) in one eye and PlGF-1 in the contralateral eye (n = 6). Retinal vessel growth area was measured in whole mounts at P5. PlGF-1–injected eyes had a mean of 41.67% ± 5.63% of the retina vascularized. Similarly, 42.18% ± 8.60% of the BSS-injected contralateral control eyes were vascularized (P = 0.91). Results are representative of two independent experiments. (b) Vessel revascularization was measured in P15 mice after induction of vessel loss by oxygen (P7–P12) followed by intravitreal injections of BSS at P13 in one eye and PlGF-1 in the contralateral eye. PlGF-1–injected eyes were 26.32% ± 2.62% vascularized; similarly, BSS-treated contralateral control eyes were 26.29% ± 2.86% vascularized (n = 6, P = 0.99). Results are representative of two independent experiments. (c) In eyes with oxygen-induced retinopathy, the mean number of vascular nuclei extending into the vitreous at P17 in ten retinal cross sections per eye (n = 8 eyes) was counted after intravitreal injections of BSS at P13 (after 5 days of 75% O2 treatment, from P7 to P12) in one eye and PlGF-1 in the contralateral eye. BSS- and PlGF-1–injected eyes showed means of 9.98 and 9.96 vascular nuclei (P = 0.61), respectively, indicating no stimulation of proliferation by PlGF-1.

Article Snippet: The sections were stained with primary antibodies against VEGFR-1 or VEGFR-2 (R&D Systems Inc.), and biotinylated Griffonia simplicifolia , thereafter G. simplicifolia , lectin I (endothelial cell specific) (Vector Laboratories Inc., Burlingame, California, USA) for 2 hours at room temperature.

Techniques: Activation Assay, Injection

Journal: CNS Neuroscience & Therapeutics

Article Title: Neuroprotective Effects of Transcranial Pulsed Current Stimulation: Modulation of Microglial Polarization in Traumatic Brain Injury

doi: 10.1111/cns.70606

Figure Lengend Snippet: The impact of tPCS on the expression of OX‐A, OX1R, MAP2, GAP43, and TGF‐β in the peri‐lesional cortex of TBI mice. (A, B) Transcriptome sequencing identified DEGs between the TBI and tPCS groups, as visualized in a volcano plot, and KEGG enrichment analysis was performed on these DEGs ( n = 3). (C–G) Representative Western blot images and quantification illustrate the levels of OX‐A, OX1R, GAP43, and MAP2 in the peri‐lesional cortex at 7 dpo ( n = 4), with β‐actin serving as the loading control. The original blots are presented in the Appendix . (H–M) Representative IF images and quantification depict the expression levels of OX1R, GAP43, and MAP2 at 7 dpo ( n = 4). (N–P) Representative IF images and quantitative analysis of NeuN and TGF‐β expression levels in the peri‐lesional cortex ( n = 4). Scale bar =100 μm. All values are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, primary antibodies against OX1R (183701AP, Proteintech, China), ZO‐1 (ab307799, Abcam, USA), MAP2 (ab254264, Abcam), GAP43 (ab277627, Abcam), TGF‐β1 (ab215715, Abcam), IBA1 (ab178846, Abcam), Bcl‐xL (ab270253, Abcam), NeuN (ab104224, Abcam), CD31 (ab182981, Abcam), BrdU (Ab6326, Abcam), CD206 (#91992, Cell Signaling Technology), and CD86 (ab119857, Abcam) were used to the sections and incubated at 4°C for 12 h. The sections were then incubated with Alexa Fluor 488 or 555‐conjugated secondary antibodies for 2 h. Fluorescence microscopy was used to capture images from three randomly selected, nonoverlapping ROIs in the cortex.

Techniques: Expressing, Sequencing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Neuroprotective Effects of Transcranial Pulsed Current Stimulation: Modulation of Microglial Polarization in Traumatic Brain Injury

doi: 10.1111/cns.70606

Figure Lengend Snippet: tPCS attenuated neuronal apoptosis and ameliorated neurological deficits via OX‐A/OX1R in TBI mice. (A–E) Motor function of TBI mice was assessed using Y‐maze, beam‐balance test, and rotarod test ( n = 8). (F–H) Representative IF images and quantitative analysis of IBA1 and OX1R expression levels in the peri‐lesional cortex ( n = 6). The arrows represent OX1R+IBA1‐positive cells. (I–K) Representative IF images and quantitative analysis of Bcl‐xL and NeuN ( n = 6). (L) Representative images of the cortical region were obtained using HE staining, Nissl staining, and transmission electron microscopy. (M) Quantification of Nissl‐stained cells ( n = 6). (N) Representative images of the cortical region were obtained using transmission electron microscopy. (O–Q) Representative Western blot bands and quantitative analysis demonstrating Bcl‐xL and NeuN levels in the peri‐lesional cortex ( n = 5), with β‐actin employed as the loading control. Scale bar = 100 μm. The original blots are presented in Appendix . All values are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, primary antibodies against OX1R (183701AP, Proteintech, China), ZO‐1 (ab307799, Abcam, USA), MAP2 (ab254264, Abcam), GAP43 (ab277627, Abcam), TGF‐β1 (ab215715, Abcam), IBA1 (ab178846, Abcam), Bcl‐xL (ab270253, Abcam), NeuN (ab104224, Abcam), CD31 (ab182981, Abcam), BrdU (Ab6326, Abcam), CD206 (#91992, Cell Signaling Technology), and CD86 (ab119857, Abcam) were used to the sections and incubated at 4°C for 12 h. The sections were then incubated with Alexa Fluor 488 or 555‐conjugated secondary antibodies for 2 h. Fluorescence microscopy was used to capture images from three randomly selected, nonoverlapping ROIs in the cortex.

Techniques: Expressing, Staining, Transmission Assay, Electron Microscopy, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Neuroprotective Effects of Transcranial Pulsed Current Stimulation: Modulation of Microglial Polarization in Traumatic Brain Injury

doi: 10.1111/cns.70606

Figure Lengend Snippet: tPCS enhances the expression of neurorepair‐related markers and neuroplasticity following TBI via the OX‐A/OX1R pathway. (A, B) Representative IF images and quantification of NeuN+BrdU‐positive cells in the peri‐lesional cortex ( n = 6). The arrows represent NeuN+BrdU‐positive cells. Scale bar = 100 μm. (C, D) Representative IF images and quantification of CD31+BrdU‐positive cells ( n = 6). The arrows represent CD31+BrdU positive cells. Scale bar = 100 μm. (E) Quantification of the brain water content ( n = 3). (F–H) Representative IF images and quantitative analysis of ZO‐1 and CD31 in the peri‐lesional cortex ( n = 6). Scale bar = 100 μm. (I, J) Representative images and quantitative analysis of Evans Blue staining at 7dpo ( n = 4). (K, L) Representative images of Golgi staining and Sholl analysis in the peri‐lesional cortex ( n = 6). The red dotted box represents the neurons selected for Sholl analysis. Scale bar =50 μm. (M, N) Representative imaging of dendrites and quantification of dendritic density ( n = 6). (O–Q) SYP and PSD95 proteins were measured and quantified at 7 dpo by Western blot ( n = 5), with β‐actin serving as a loading control. The original blots are presented in Appendix . (R, S) Representative TEM images and quantification of postsynaptic densities in the perilesional cortex. Scale bar = 1 μm. All values are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, primary antibodies against OX1R (183701AP, Proteintech, China), ZO‐1 (ab307799, Abcam, USA), MAP2 (ab254264, Abcam), GAP43 (ab277627, Abcam), TGF‐β1 (ab215715, Abcam), IBA1 (ab178846, Abcam), Bcl‐xL (ab270253, Abcam), NeuN (ab104224, Abcam), CD31 (ab182981, Abcam), BrdU (Ab6326, Abcam), CD206 (#91992, Cell Signaling Technology), and CD86 (ab119857, Abcam) were used to the sections and incubated at 4°C for 12 h. The sections were then incubated with Alexa Fluor 488 or 555‐conjugated secondary antibodies for 2 h. Fluorescence microscopy was used to capture images from three randomly selected, nonoverlapping ROIs in the cortex.

Techniques: Expressing, Staining, Imaging, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Neuroprotective Effects of Transcranial Pulsed Current Stimulation: Modulation of Microglial Polarization in Traumatic Brain Injury

doi: 10.1111/cns.70606

Figure Lengend Snippet: Treatment with tPCS modulates M1/M2 microglia polarization via OX‐A/OX1R‐mediated NF‐κB pathway following TBI. (A, B) Representative IF images and quantification of CD86+IBA1‐positive cells in the peri‐lesional cortex ( n = 6). (C, D) Representative IF image and quantification of CD206+IBA1‐positive cells ( n = 6). (E–G) Representative immunofluorescence image and quantification of p65+IBA1‐positive cells ( n = 6). (H, I) Representative IF image and quantitative analysis of NeuN and TGF‐β ( n = 6). (J–N) Representative Western blot bands and quantification showing levels of phosphorylated (p)‐p65, p‐IκBα, iNOS, and Arg‐1 proteins in the peri‐lesional cortex ( n = 5), with β‐actin serving as a loading control. All values are mean ± SD. Scale bar = 100 μm. The original blots are presented in Appendix . * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, primary antibodies against OX1R (183701AP, Proteintech, China), ZO‐1 (ab307799, Abcam, USA), MAP2 (ab254264, Abcam), GAP43 (ab277627, Abcam), TGF‐β1 (ab215715, Abcam), IBA1 (ab178846, Abcam), Bcl‐xL (ab270253, Abcam), NeuN (ab104224, Abcam), CD31 (ab182981, Abcam), BrdU (Ab6326, Abcam), CD206 (#91992, Cell Signaling Technology), and CD86 (ab119857, Abcam) were used to the sections and incubated at 4°C for 12 h. The sections were then incubated with Alexa Fluor 488 or 555‐conjugated secondary antibodies for 2 h. Fluorescence microscopy was used to capture images from three randomly selected, nonoverlapping ROIs in the cortex.

Techniques: Immunofluorescence, Western Blot, Control

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) All three members of human TIM family interact with NUR77 as shown by coimmunoprecipitation assay. N=3 independent experiments (B) Top panel: Domain architecture of TIM-1. Middle Panel: Coimmunoprecipitation experiment demonstrating that only the entire extracellular domain (EED) and IgV domain (IgVD) of TIM-1 interact with NUR77 and not the mucin (MD) or the cytoplasmic domain (CD). Bottom panel: long exposure of the input blot showing the CD protein band. N=2 independent experiments. (C) The transactivation function of NUR77 is repressed by all three human TIM proteins in a dose dependent manner (Top panel). Full length TIM-1 protein is required for repression of NUR77 transcriptional activity (Bottom panel). Data from at least three independent experiments is presented. (D) Binding of NUR77 to its response elements (NBRE) is abrogated by the full length TIM proteins in an electrophoretic mobility shift assay using p32 labeled NBRE oligo. Competition with unlabelled probe (cold competition) reveals the specificity of NUR77 binding. A representative autoradiograph is shown (left) and the relative amount of NUR77 protein binding to NBRE probe was quantified from three independent experiments (right). ***, P<0.001, **, P<0.01, *,P<0.05.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Co-Immunoprecipitation Assay, Activity Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Autoradiography, Protein Binding

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) Quantitative RT-PCR measurement of NUR77 transcript induced by PMA and ionomycin treatment in control and TIM-1 shRNA stable knockdown clones of 769-P cells (N=3 experiments). (B) Western analysis of the protein samples from (A) indicating the decrease in NUR77 abundance in the presence of TIM-1. Con, control. N= 3 independent experiments. (C) Overexpression of TIM-1 in HK-2 cells resulted in decreased NUR77 protein abundance induced by desferrioxamine (Des) (compare lanes: 3 and 4), which is perturbed by chloroquine treatment (CQ) (Lanes: 7 and 8). N=3 independent experiments. (D) Silencing of TIM-1 expression in HK-2 cells resulted in increased cell death in an in vitro epithelial cell injury model induced by a combination of ATP and glucose depletion and calcium overload. N= 3 independent experiments. IRI, Ischemia reperfusion injury (E) Confocal microscopy analysis of the localization pattern of endogenous hTIM-1 (green) in 769-P cells (Top panel) and GFP-tagged hTIM-1 stably expressed in Jurkat cells (Bottom panel). Scale bar, 10 µm. N=2 experiments. (F) Colocalization of ectopically expressed TIM-1 (green) in Cos-7 cells with markers of early endosomes (EEA1), Golgi complex (Giantin), and lysosomes (Lamp2a). Scale bar, 5 µm. Colocalization coefficients: hTIM-1/EEA1:0.34±0.06; hTIM-1/Giantin: 0.74±0.04; hTIM-1/Lamp2a: 0.55±0.08; n=5 cells each from two independent experiments (G). Colocalization of TIM-1 (blue) and ectopically expressed NUR77 (green) with lysosomes (Lysotracker red) in 769-P cells. Colocalization coefficients: hTim-1/NUR77: 0.59±0.12; hTIM-1/Lysotracker: 0.47±0.09; NUR77/Lysotracker: 0.51±0.08; and HK-2 cells; Colocalization coefficients: hTim-1/NUR77: 0.52+−.06; hTIM-1/Lysotracker: 0.56+0.08; NUR77/Lysotracker: 0.62+0.07; n=5 cells each from two independent experiments. Scale bar, 10 µm.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Quantitative RT-PCR, shRNA, Clone Assay, Western Blot, Over Expression, Expressing, In Vitro, Confocal Microscopy, Stable Transfection

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) Left panel: Mutations in the MILIBS of TIM-1 abrogated TIM-1-mediated degradation of NUR77 protein (top). The double mutant of cytoplasmic tyrosine residues Y299 and Y335 partially restored the protein abundance of NUR77, but did not affect that of GFP (bottom). Right panel: Quantitation of NUR77 and GFP abundance in the presence of wild type and mutant TIM-1 based on three independent experiments. (B) Confocal microscopy analysis of the localization pattern of wild type, MILIBS, cytoplasmic tyrosine residue mutants of TIM-1, revealing increased cell surface localization of the MILIBS TIM-1 mutants. Scale bar, 5 µm (Left). Quantification of the localization of wild-type TIM-1 and the MILIBS mutant. N=10 cells each from three independent experiments (Right). (C) Inability of MILIBS mutants to mediate NUR77 degradation does not arise from lack of interaction with NUR77: Wild type and MILIBS mutant TIM-1 interact with NUR77 with equal efficiency demonstrated by GST pull down assay (Middle panel). IgV and mucin domains served as positive and negative controls of the interaction between TIM-1 and NUR77. Shown is a representative of three independent experiments.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Mutagenesis, Quantitation Assay, Confocal Microscopy, Pull Down Assay

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) A form of TIM-1 lacking the signal peptide deleted TIM-1 is not glycosylated and unable to mediate degradation of NUR77. N= 2 independent experiments. (B) Perturbation of endocytic pathway using a dominant negative Dynamin construct (Dyn2K44A) did not affect TIM-1 mediated degradation of NUR77. N= 3 independent experiments (Left panel) and quantitation of protein abundance based on three independent experiments (Right panel). (C) Dominant negative constructs of Eps15 (DIII and D3delta2) substantially abrogated TIM-1 mediated degradation of NUR77. N=3 independent experiments (left panel). NUR77 protein abundance was quantified based on three independent experiments (right panel). (D) TIM-1 is constitutively endocytosed as revealed by the increased cell surface localization of transiently transfected TIM-1 in 293T cells, upon blockade of endocytosis using dominant negative constructs of dynamin-2 and Eps15 as assayed by flow cytometry. Perturbation of clathrin vesicle formation further enhanced the increased cell surface localization of the MILIBS mutant. Shaded histogram represents the control antibody staining and the open histogram represents hTIM-1 staining. Two peaks of TIM-1 staining reveal the dynamic cycling of TIM-1. N= 2 independent experiments.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Dominant Negative Mutation, Construct, Quantitation Assay, Transfection, Flow Cytometry, Mutagenesis, Staining

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) Confocal immunofluorescence analysis of TIM-1 (green) colocalization with Clathrin (Top panel) and Caveolin-1 (Bottom panel) where yellow indicates colocalization. Scale bar, 10 µm. Colocalization coefficients: hTIM-1/Clathrin: 0.68±0.05; hTIM-1/Caveoli: 0.12±0.04; n=5 cells each from two independent experiments. (B). Retrograde translocation of TIM-1(green) from cell surface to lysosomes and endoplasmic reticulum through the trans-Golgi network in a clathrin-mediated pathway demonstrated by endocytosis assay; subcellular organelle markers are in red. Shown is a representative image. Scale bar, 10 µm. Colocalization coefficients: hTIM-1/EEA1: 0.57±0.05; hTIM-1/p230: 0.35±0.08; hTIM-1/Lamp2a (30 min): 0.46±0.09; hTIM-1/Clathrin: 0.38±0.06; hTIM-1/Lamp2a (60 min): 0.55±0.03; hTIM-1/Calnexin: 0.31±0.03; n=5 cells each from two independent experiments. (C to E) Model depicting the clathrin-mediated constitutive trafficking of TIM-1 and the phenomenon of TIM-1 mediated NUR77 degradation. TIM-1 and other TIM family members (red) are targeted to cell surface (C), prior to constitutive endocytosis through clathrin-coated vesicles (D). Following fusion with the trans-golgi network, TIM-1 undergoes retrograde translocation to endoplasmic reticulum (ER) and lysosomes (purple circles). TIM-1 might come in contact with NUR77 possibly in ER or vesicles leading to recruitment of this protein complex to PS-rich endosomes and lysosomes depending on the TIM-1-PS interaction, facilitating sorting of NUR77 to lysosomes and ultimately resulting in the degradation of NUR77 by lysosomal enzymes. TIM-1 is presumably retained in the vesicles (E).

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Immunofluorescence, Translocation Assay, Endocytosis Assay

Journal: Nature Communications

Article Title: Heparan sulfate proteoglycans present PCSK9 to the LDL receptor

doi: 10.1038/s41467-017-00568-7

Figure Lengend Snippet: Enzymatic removal of liver heparan sulfate releases PCSK9 and ablates its activity. a , b Infusion of heparinase I prior to the injection of PCSK9 (10 µg) completely inhibits PCSK9-induced degradation of LDLR. Western blot of representative samples is shown in a and quantification of LDLR in b (control n = 7, PCSK9 n = 6, heparinase n = 5, heparinase/PCSK9 n = 5). Heparinase treatment unmasks the antigenicity of the major liver HSPG syndecan-1 ( middle panel ). Beta-actin is used as loading control ( lower panel ). c Heparinase I treatment leads to an increase in plasma PCSK9 as measured by ELISA 15 min after injection (control n = 6, heparinase n = 6). d Western blot (representative samples) of liver syndecan-1 is used as control of heparinase injection. Beta-actin is shown as loading control. e – h Transgenic mice with constitutive expression of human heparanase ( Hpa -tg) ( n = 7) have increased plasma PCSK9 ( e ), increased liver LDLR ( f , g ), and reduced plasma cholesterol ( h ) compared to control WT mice ( n = 6). Bar graphs show mean with s.e.m. error bars. Statistical significance was evaluated using a two-tailed Student’s t -test. Supplementary Fig. shows uncropped gel images

Article Snippet: The following antibodies were used for Western blotting: anti-LDLR (Abcam ab52818, 1:1000), anti-GAPDH (Sigma Aldrich G8795, 1:2000), anti-beta actin (Sigma A5441, 1:5000), anti-PCSK9 (R&D Systems AF3985, 1:1000), anti-syndecan-1 (R&D AF3190, 1:1000), and immunostainings: anti-PCSK9 (R&D Systems AF3888, 2 µg/ml), and fluorescent secondary antibodies (Invitrogen).

Techniques: Activity Assay, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Expressing, Two Tailed Test