Journal: Cureus

Article Title: Melittin Alleviates Oxidative Stress Injury in Schwann Cells by Targeting Interleukin-1 Receptor Type 1 to Downregulate Nuclear Factor Kappa B-Mediated Inflammatory Response In Vitro

doi: 10.7759/cureus.65721

Figure Lengend Snippet: Primer sequences

Article Snippet: Each PVDF membrane was closed with 5% skim milk (BD, Franklin Lakes, NJ) at room temperature for 60 min and then incubated overnight with the following primary antibodies at 4°C: anti-HIF‐1α antibody (#14179, Cell Signaling, 1:1000), anti-GAPDH antibody (ab245355, Abcam, 1:1000), anti-VEGF antibody (#9698, Cell Signaling, 1:1000), anti-IL-1R1 antibody (#6775, Cell Signaling, 1:1000), anti-ENO1 antibody (#3810S, Cell Signaling, 1:1000), anti-C-JUN antibody (#9165T, Cell Signaling, 1:1000), anti-glial cell line-derived neurotrophic factor (GDNF) antibody (#47808, Cell Signaling, 1:1000), anti-IRAK1 antibody (#4395, Cell Signaling, 1:1000), anti-IKKα antibody (#2682 Cell Signaling, 1:1000), anti-p65 antibody (ab16502, Abcam, 1:800), anti-p50 antibody (ab305263, Abcam, 1:900), anti-IKB-α antibody (#8943, Cell Signaling, 1:900), anti-NGF antibody (#2046, Cell Signaling, 1:1000), and anti-β-actin antibody (ab179467, Abcam, 1:1000).

Techniques:

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Steady-state distribution and delivery to the plasma membrane of PLAP and NTR-PLAP in transfected FRT cells. (A) Schemes of PLAP and NTR-PLAP (a fusion protein containing the ectodomain of p75NTR and the C-terminal GPI attachment signal of PLAP). (B) FRT cells stably expressing PLAP or NTR-PLAP were grown to confluence and pulsed overnight with [35S]met–cys or [35S]cys, respectively. After surface biotinylation from the apical (Ap) and basolateral (BL) domains, cells were lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (C) FRT cells were labeled for 20 min with [35S]met–cys or [35S]cys. At different times of the chase, surface proteins were biotinylated from the apical (Ap) or the basolateral (BL) side. Cells were then lysed and immunoprecipitated with antibodies against PLAP or NTR and with streptavidin beads. Samples were run on 10% SDS-PAGE and detected by fluorography. PLAP and NTR-PLAP are directly sorted to the apical surface in FRT cells, whereas a small percentage of the two proteins is delivered with similar kinetics to the basolateral domain.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Transfection, Stable Transfection, Expressing, Immunoprecipitation, Labeling, SDS Page

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Pulse-chase analysis and purification on sucrose gradients of transmembrane forms of PLAP and p75NTR in transfected FRT cells. (A) Schemes of p75NTR and PLAP-PS321 (a fusion protein containing the ectodomain of PLAP and the transmembrane and the cytoplasmic domain of the PS321 NTR mutant). (B) Pulse-chase and TX-100 extraction of p75NTR and PLAP-PS321. FRT cells expressing p75NTR and PLAP-PS321 were labeled with [35S]cys or [35S]met–cys, respectively, and subsequently treated as described in Figure ​Figure2A.2A. (C) Purification of p75NTR- and PLAP-PS321–enriched fractions on sucrose density gradients. Labeled proteins of FRT cells expressing p75NTR and PLAP-PS321 were solubilized in TX-100 and purified as described in Figure ​Figure2B.2B. Both transmembrane proteins are soluble in TX-100 and do not float on sucrose density gradients.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Pulse Chase, Purification, Transfection, Mutagenesis, Expressing, Labeling

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored, secretory, and transmembrane proteins after FB1 treatment. (A) FRT cells stably expressing PLAP, NTR-PLAP, or gD1-DAF were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After 30 min of labeling with [35S]met–cys or [35S]cys and a 2-h chase, cells were surface biotinylated from the apical (Ap) or the basolateral (BL) domain, lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (B) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. Apical PLAP and NTR-PLAP, but not basolateral gD1-DAF, became unpolarized after FB1 treatment. (C) FRT cells expressing PLAP-sec, NTR-sec, p75NTR, or PLAP-PS321 were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After labeling with [35S]met–cys or [35S]cys for 20 min and a chase of 2 h, apical and basolateral media were collected separately and cells were surface biotinylated from apical or basolateral domains. Polarized secretion of the secretory forms was revealed by immunoprecipitation in the apical and basolateral medium (top two panels), whereas biotinylated transmembrane proteins were revealed by double immunoprecipitation with specific antibody and streptavidin beads (bottom two panels). (D) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. FB1 does not alter the sorting of transmembrane and secretory proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Stable Transfection, Labeling, Immunoprecipitation

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored and transmembrane proteins after cholesterol depletion. (A) FRT cells expressing PLAP, NTR-PLAP, gD1-DAF, p75NTR, or PLAP-PS321 were grown on filters and treated with mevinolin (Mev.; 10 μM) for 48 h and M-β-CD (10 mM) for 60 min. Cells were then pulse labeled for 30 min, chased for 2 h, and biotinylated as usual. (B) Amounts of labeled proteins were quantified in three independent experiments and are shown as means ± SD. Hatched bars represent apical (Ap) proteins, and black bars represent basolateral (BL) proteins. Removal of cholesterol does not alter the surface expression of apical and basolateral GPI-anchored and transmembrane proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Labeling

Journal: ESC Heart Failure

Article Title: The influence of inhibiting renal neural regeneration on the efficacy of renal denervation to chronic heart failure

doi: 10.1002/ehf2.13655

Figure Lengend Snippet: Comparisons of plasma parameters and mRNA expression of indicator of nerve regeneration in the renal arteries among the five groups. The levels of plasma renin (A), noradrenaline (B), Nog‐B (C), N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) (D) and the ratio of heart to body weight (E) in the control, heart failure (HF), renal denervation (RDN), Nog and NEP groups. The relative mRNA levels of calcitonin gene‐related peptide (CGRP) (F), nerve growth factor (β‐NGF) (G), and growth‐associated protein 43 (GAP‐43) (H). * P < 0.05 vs. the HF and # P < 0.05 vs. the RDN group.

Article Snippet: After blocking with 5% non‐fat milk, membranes were incubated with the following primary antibodies: CGRP polyclonal antibody (1:8000; Sigma‐Aldrich, USA; Catalog C8198), β‐NGF (1:1000; R&D Systems Inc., USA; Catalog AF‐556‐NA), GAP‐43 (1:500; BIOSS, China; Catalog BS‐0154R), or β‐actin (1:1000; Boster, China; Catalog BM0627; Clone AC‐15).

Techniques: Clinical Proteomics, Expressing, Control

Journal: ESC Heart Failure

Article Title: The influence of inhibiting renal neural regeneration on the efficacy of renal denervation to chronic heart failure

doi: 10.1002/ehf2.13655

Figure Lengend Snippet: Comparisons of plasma parameters, heart weight, body weight, and relative mRNA expression of indicator of nerve regeneration in the renal arteries among the five groups

Article Snippet: After blocking with 5% non‐fat milk, membranes were incubated with the following primary antibodies: CGRP polyclonal antibody (1:8000; Sigma‐Aldrich, USA; Catalog C8198), β‐NGF (1:1000; R&D Systems Inc., USA; Catalog AF‐556‐NA), GAP‐43 (1:500; BIOSS, China; Catalog BS‐0154R), or β‐actin (1:1000; Boster, China; Catalog BM0627; Clone AC‐15).

Techniques: Clinical Proteomics, Expressing, Control

Journal: ESC Heart Failure

Article Title: The influence of inhibiting renal neural regeneration on the efficacy of renal denervation to chronic heart failure

doi: 10.1002/ehf2.13655

Figure Lengend Snippet: The protein expression of calcitonin gene‐related peptide (CGRP), nerve growth factor (β‐NGF) and growth‐associated protein 43 (GAP‐43) in the renal arteries in the different groups. Western blot analysis of CGRP, GAP‐43 and β‐NGF expression in the control (A), heart failure (HF) (B), renal denervation (RDN) (C), Nog (D) and NEP (E) groups and relative mRNA levels of CGRP (F), β‐NGF (G), and GAP‐43 (H) in the renal arteries in the different groups. * P < 0.05 vs. the HF and # P < 0.05 vs. the RDN group.

Article Snippet: After blocking with 5% non‐fat milk, membranes were incubated with the following primary antibodies: CGRP polyclonal antibody (1:8000; Sigma‐Aldrich, USA; Catalog C8198), β‐NGF (1:1000; R&D Systems Inc., USA; Catalog AF‐556‐NA), GAP‐43 (1:500; BIOSS, China; Catalog BS‐0154R), or β‐actin (1:1000; Boster, China; Catalog BM0627; Clone AC‐15).

Techniques: Expressing, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.1 method. (A) Colony of human iris-derived iPS cells. (B-D) Cell colony in differentiation culture: (B) 6th day in STEP-1 medium, (C) 18th day in STEP-2 medium, and (D) 25th day in STEP-3 medium. Scale bar in (A-D), 100 µm. (E) Fold-increase in p75NTR mRNA expression level at different steps relative to STEP-0: * P<0.05. (F) Cell morphology on 35th day of differentiation in STEP-3 medium. (G) Immunostaining for αA-crystallin in the same area as indicated in (F). (H) Immunostaining for SOX2 in the same area as in (G). (I) Merged image of αA-crystallin and SOX2 immunostaining. Scale bar in (F-I), 100 µm. Ex, experiment; iPS cells, induced pluripotent stem cells.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Derivative Assay, Expressing, Immunostaining

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Changes in gene expression levels at each step of differentiation induction using Ex.4 the method. (A) SOX2 , (B) PAX6 , (C) p75NTR , (D) αA-crystallin , (E) type-IV collagen ; fold-changes are relative to gene expression level in STEP-0: * P<0.05, ** P<0.01, *** P<0.005. Ex, experiment.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.4 method. (A) Gross observation of cell aggregate. Reduced opacity is observed in certain areas. Scale bar, 2 mm. (B) H&E-stained specimen of cell aggregate. Scale bar, 200 µm. (C) Enlarged view of H&E staining in Area 1 of the specimen in (B). Cells displaying epithelial-like morphology have infiltrated inside the cell aggregate (yellow arrowhead). (D-G and I-L) Immunostaining of cell aggregates for (D and I) SOX2, (E and J) p75NTR, (F and K) αA-crystallin, and (G and L) type IV collagen. Blue in immunostaining images: DAPI staining of nuclei. (H) Enlarged view of H&E staining of Area 2 in the specimen in (B), where the cells on the surface of the aggregate have transitioned from a multilayer to a monolayer (white arrowhead). Scale bar in (C-L), 50 µm. Ex, experiment; H&E, hematoxylin and eosin.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: BoNT/A blocked NGF-induced CGRP release and -enhancement of 20 nM CAP-evoked CGRP release. ( A ) After 2 days in the presence of 50 ng/mL NGF, TGNs were starved of the neurotrophin as detailed before, without or with the inclusion of 100 nM BoNT/A during this latter step. The release experiment was performed as described in A. ( B ) At the end of the protocol, one well each of BoNT/A-treated and non-treated cells were solubilised in 1× LDS and subjected to Western blotting. PVDF membranes were cut horizontally midway between the 25 k and 37 k molecular weight markers. The upper portion was exposed to antibodies reactive with syntaxin-1 (mouse monoclonal, 1:2000) and the lower piece probed with an antibody recognising both intact and BoNT/A-truncated SNAP-25 (mouse monoclonal, 1:3000). ( C ) The amount of cleaved SNAP-25 in BoNT/A-treated cells was calculated as a % (mean + s.e.m., N = 3) of the total SNAP-25 (intact + BoNT/A product). ( D ) Total CGRP (pg/well). ( E – G ) Histograms showing: ( E ) spontaneous CGRP release during the second 30 min. incubation into HBS without (grey bars) or induced by 100 ng/mL NGF (blue bars), ( F ) during the third period evoked by 20 nM CAP (minus the spontaneous release), and ( G ) during the third incubation with 1 µM CAP minus the spontaneous release. In all cases, CGRP release is expressed as a % of total CGRP (mean + s.e.m., N = 3, n = 9). Asterisks summarise the results of unpaired one-tailed Welch tests applied to the data plotted in panels E, F, and G, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Western Blot, Molecular Weight, Incubation, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: Chimera/EA effectively inhibits 1 µM CAP-evoked CGRP release from starved TGNs, and its enhancement by NGF. TGNs were starved and incubated with 100 nM/EA using a protocol identical to that described previously for BoNT/A ( A). ( A ) Western blotting with anti-SNAP-25 antibodies confirms the disappearance of intact SNAP-25 and the appearance of a much faster-migrating product in cells exposed to/EA (+) but not control (−). ( B ) Histogram displaying the % of SNAP-25 cleaved, which was calculated as in C. ( C ) Total amounts (pg/well) of CGRP, determined as before, in control and/EA-treated cells. ( D ) Amounts of spontaneous CGRP release (% total) into HBS only, (grey bars), and that during incubation with 100 ng/mL NGF (blue bars), and ( E ) upon stimulation with 1 µM CAP (minus the spontaneous release) (mean + s.e.m. N = 3, n = 9). Unpaired one-tailed Welch test was applied to the data plotted in panels ( C – E ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Western Blot, Control, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: NGF induces a minor increase in Ca 2+ - and SNARE-dependent CGRP release, whereas it greatly enhances the CAP-evoked exocytosis which is blocked by BoNT/A and/EA at low [CAP] but only abolished by BoNT/EA at higher [CAP]. ( A ) Illustrates the effect of acute NGF on CGRP exocytosis from control neonatal rat TGNs starved of the neurotrophin for 2 days, and ( B ) in TGNs pre-treated with BoNT/A or /EA. ( A ) NGF binds to its receptor TrkA, activates the signalling cascades shown , and induces Ca 2+ influx by an unidentified mechanism ( ? ). Elevated intracellular Ca 2+ ([Ca 2+ ] i ) triggers the fusion of large dense core vesicles (LDCVs) via SNARE-complexes (VAMP, syntaxin-1 and SNAP-25), thereby, causing exocytotic release of CGRP and surface delivery of vesicle constituents. This acute potentiation by NGF can involve the phosphatidylinositol 3-kinase—Src (PI3K-Src) pathway, which promotes trafficking of LDCVs, and insertion of their TRPV1 channels into the plasmalemma by Ca 2+ -regulated exocytosis (blue arrows) c.f. [ , ]. Additionally, the phospholipase C γ (PLCγ) cascade leads to sensitisation of TRPV1 already on the plasmalemma (red dashed arrows) . The outcome of these composite influences of NGF on TRPV1 is that when the channel is activated by CAP [Ca 2+ ] I is raised even more than normally [ , , ] and this further enhances CGRP release . ( B ) The proteases of BoNT/A and/EA delete 9 (purple arrow) and 26 (green arrow) residues from SNAP-25 (Insert), respectively, preventing the fusion of LDCVs; this blocks the minimal CGRP exocytosis elicited by NGF (arrow with crosses, left) and its enhancement of the release evoked by 20 nM CAP ( ) (arrow with crosses, centre). Stronger stimulation of TRPV1 with 1 µM CAP ( ) induces a lot more Ca 2+ influx ( [Ca 2+ ] i , ( [Ca 2+ ] i , ( [Ca 2+ ] i ; ) which causes a moderate increase in CGRP release but overcomes the inhibition by BoNT/A (purple cross with broken lines, right) while/EA (green cross, right) remains effective in diminishing CGRP release. Acute sensitisation by NGF of TRPV1 selectively enhances neuropeptide exocytosis stimulated by low [CAP] (<100 nM) and only moderately affects responses to ≥100 nM CAP ( B). Despite being impotent against 1 µM CAP-evoked CGRP release in starved cells (( B ), , and G), BoNT/A partially inhibits the moderate NGF-enhancement of 1 µM CAP-evoked CGRP release ( G), implicating membrane trafficking in the sensitisation process (B, PI3K-Src stimulated pathway) in accordance with its inhibition of NGF-induced, Ca 2+ -dependent CGRP exocytosis ( E).

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Control, Inhibition, Membrane