anti nbce1  (Alomone Labs)

Journal: bioRxiv

Article Title: Functional contribution of astrocytic Kir4.1 channels to spasticity after spinal cord injury

doi: 10.1101/2024.10.11.617793

Figure Lengend Snippet: (A) NBCe1 pseudo-gel images from capillary western blot of lumbar segment from CTL (left, black) and SCI (right, red) mice. (B) Group mean quantification of the NBCe1 band (∼140 kDa) normalized to CTL group (CTL: n=4 mice; SCI: n=7 mice). (C) Two-photon fluorescence images of lumbar cord showing identified astrocytes (tdTomato) and cells loaded with pH-sensitive BCECF. Scale bar: 50 µm; inset: BCECF-loaded astrocytes (white arrows). Scale bar : 15 µm. (D) Pseudocolor images of BCECF signals in tdTomato(+) astrocytes before (left) and after 40s of L-glutamate application (right). ROI#1 shows increased pixel intensity, while ROIs #2 and #3 show decreased signal intensity in response to L-glutamate. (E) BCECF signal changes in response to L-glutamate in astrocytic ROIs from the same FOV in (D), showing alkalinization (top) or acidification (bottom). (F) Percentage of alkalinizing and acidifying responses to glutamate in astrocytes from CTL (top, black) and SCI (bottom, red) mice (CTL: n=263 ROIs, 4 mice; SCI: n=172 ROIs, 4 mice). (G) (Top) Average astrocyte acidic responses during L-glutamate exposure in aCSF showing enhanced acidosis post-SCI; (Bottom) Reduced response with NBCe1 blocker, S0859 (100 µM). (H) Violin plots of maximum BCECF acidic changes during L-glutamate exposure in CTL (left, black) and SCI (right, red) mice without (left) or with (right) S0859 (CTL: n=158 ROIs, 4 mice; SCI: n=139 ROIs, 4 mice; with S0859, CTL: n=64 ROIs, 4 mice; SCI: n=32 ROIs, 3 mice). Data: mean ± S.E.M, hatched line in violin plots are median. ns: not significant, *P < 0.05, ***P < 0.001. Mann-Whitney test for B; Kruskal-Wallis one-way ANOVA followed by Dunn’s post-hoc test for H; Fisher’s test for F. See also Figure S3.

Article Snippet: Samples were probed with a rabbit polyclonal Kir4.1 antibody (1:60; APC-035, Alomone, Israel) or a rabbit polyclonal NBCe1 antibody (1:90; ANT-075, Alomone, Israel) and revealed with the appropriate detection module (DM-001, ProteinSimple, Bio-Techne, MN).

Techniques: Western Blot, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional contribution of astrocytic Kir4.1 channels to spasticity after spinal cord injury

doi: 10.1101/2024.10.11.617793

Figure Lengend Snippet: (A) Confocal images showing endogenous GFP (A1), and immunofluorescent staining for Kir4.1 (A2), NeuN (A3), and merged image (A4) in the ventro-lateral lumbar cord of an Aldh1L1-eGFP mouse. Scale bar: 120 µm. Higher magnification (middle) shows astrocytes (A1’) expressing Kir4.1 (A2’) among neurons (A3’). Scale bar: 50 µm. Insets: close-up of astrocytes (A1’’) expressing Kir4.1 (A2’’) enwrapping a large ventro-lateral neuron, likely motoneurons (A3’’). Scale bar: 10 µm. GM: gray matter; WM: white matter. (B) Superimposed Ba 2+ -sensitive currents (top) from GFP+ astrocytes clamped at RMP in response to voltage pulses (bottom) in CTL mice with classic (pH=7.4, left) and acidic (pH=6.9, right) intracellular solution. (C) Superimposed Ba 2+ -sensitive currents (top) from a GFP+ astrocyte at RMP with voltage pulses (bottom) in an SCI mouse without (left) and with (right) NBCe1 blocker, S0859. (D) I/V plots of Ba 2+ -sensitive currents in CTL (black), CTL acidosis (yellow), SCI (red), and SCI + S0859 (blue). (E) Peak amplitude at -140 mV from Ba 2+ -sensitive currents (CTL: n=23 astrocytes, 5 mice; CTL pH acidosis: n=14 astrocytes, 4 mice; SCI: n=25 astrocytes, 5 mice; SCI + S0859: n=18 astrocytes, 5 mice). (F) Experimental design diagram for recording K + -inward currents in astrocytes. (G) Typical K + -inward current traces following a K + puff (12mM) in astrocytes from CTL mice without (black) and with (yellow) acidic solution, and SCI mice without (red) and with (blue) S0859. (H) Peak amplitude of K + puff-mediated currents (CTL: n=14 astrocytes, 5 mice; CTL pH acidosis: n=12 astrocytes, 4 mice; SCI: n=14 astrocytes, 5 mice; SCI + S0859: n=12 astrocytes, 4 mice). Data: mean ± S.E.M. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis one-way ANOVA followed by Dunn’s post-hoc test for E and H. See also Figure S4.

Article Snippet: Samples were probed with a rabbit polyclonal Kir4.1 antibody (1:60; APC-035, Alomone, Israel) or a rabbit polyclonal NBCe1 antibody (1:90; ANT-075, Alomone, Israel) and revealed with the appropriate detection module (DM-001, ProteinSimple, Bio-Techne, MN).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Airway succinate chemosensing induces CFTR-dependent anion secretion and mucus clearance which is impaired in cystic fibrosis

doi: 10.1101/2024.03.26.586799

Figure Lengend Snippet: (A) M3 muscarinic receptor staining (M3r) is excluded from ciliated cells as indicated by the red arrowheads and acetylated tubulin staining (Ac-Tub), but co-localizes with CC10 staining in secretory or club cells indicated by white arrowheads. (B) SLC4A4 co-localizes with M3r staining as indicated with the white arrow heads. (C) EGFP positive cell showing M3r staining. Representative image of 5 animals per group. Bars are 50 µm.

Article Snippet: For colocalization 1:100 anti-CHRM3 was incubated with 1:500 anti-Clara Cell Secretory Protein (CCSP; Merck Millipore cat#07– 623), 1:200 anti-alpha tubulin (Santa Cruz, cat#sc-5286) or 1:100 anti-NBCe1 (Alomone, ANT-075,) overnight at 4 °C, and incubated with secondary antibody 1:1,000 Alexa fluor 555 goat anti Rabbit IgG (H+L) (Life Technologies, cat#A-A21428), 1:1,000 Alexa fluor 568 goat anti Mouse IgG2b (H+L) (Life Technologies, cat#A-A21144) or 1:1,000 Alexa fluor 555 goat anti Rabbit IgG (H+L) (Life Technologies, cat#A-A21428) respectively.

Techniques: Staining

Journal: Science signaling

Article Title: VPS34-dependent control of apical membrane function of proximal tubule cells and nutrient recovery by the kidney

doi: 10.1126/scisignal.abo7940

Figure Lengend Snippet: (A) Volcano plot analysis of protein abundance in kidney cortex from Vps34ΔPT and control mice. The log-p value of a two-tailed t-test is plotted against the log2 ratio of Vps34ΔPT/control. The proteins beyond the lines indicate significance after correction for multiple testing. Proteins are related to retromer complex (green), endocytic proteins (pink), autophagy (blue), proteasome (black) and others (red). N=4 mice/group. (B and C) Western blot of kidney cortex from Vps34ΔPT and control mice (B) for the proximal tubule glucose transporter SGLT2, the sodium bicarbonate transporter NBC1 and the amino acid transporters B0AT1, 4F2hc, y+Lat1, and γ+Lat2. Each lane represents an individual mouse. Densitometry (C) of the Western blots in (B). n = 6 mice/group, *P <0.005 compared to control. (D) Immunohistochemical staining of kidney cortex from Vps34ΔPT mice for SGLT2, NBC1, γ+Lat1 or γ+Lat2 (green) and Alexa555-lactoglobulin (red). Alexa647-phalloidin (blue) was used to mark actin filaments of the brush border membrane to identify proximal tubules. White bars indicate the border between proximal tubular cells with Vps34 and that were positive for Alexa555-labeled lactoglobulin uptake (asterisk) and cells deficient in Vps34 and that were devoid of Alexa555-labeled lactoglobulin (hash). Images are representative of 2 sections from 4 mice. Scale bar = 20 μm. (E) Volcano plot of proteins with altered abundance in kidney cortex from Vps34ΔPT mice. Antiviral defense proteins, IFN response proteins, and SARS-Cov-2 receptors are indicated. N=4 mice/group. (F) Western blot of kidney cortex from Vps34ΔPT and control mice for ACE2. Each lane represents an individual mouse. Graph shows densitometry of the Western blot. n = 6 mice/group, #P < 0.001 compared to control (the β-actin Western blot from Figure 2B is also the loading control for this figure panel). (G) Immunohistochemical staining of kidney cortex from Vps34ΔPT mice for ACE2 (green) and Alexa555-lactoglobulin (red). Alexa647-phalloidin (blue) was used to mark actin filaments of the brush border membrane to identify proximal tubules. White bars indicate the border between proximal tubular cells with Vps34 and that were positive for Alexa555-labeled lactoglobulin uptake (asterisk) and cells deficient in Vps34 and that were devoid of Alexa555-labeled lactoglobulin (hash). Images are representative of 2 sections from 4 mice. Scale bar = 20 μm.

Article Snippet: The following antibodies were used: rabbit anti-Vps34 (Immobilon); guinea pig anti-megalin ( 81 ); rabbit anti-clathrin (Abcam, Cambridge UK, ab 21679); rabbit anti-EEA1 and rabbit anti-Rab11 both purchased from Invitrogen, rat anti-Lamp2 (sc-19991, Santa Cruz Biotechnology), mouse-anti NKA (MerckMillipore, Germany); rabbit anti-NBC1 (Alomone labs), rabbit and guinea pig anti-SGLT2 (H. Köpsell, University Würzburg), rabbit anti-S6P (5G10), rabbit anti-phospho-S6P (D57.2.2E), both purchased from Cell Signaling Technologies; sheep anti-NDRG1, sheep anti-phospho-NDRG1 (gift of D. Alessi, University of Dundee, UK); rabbit anti-B 0 AT1 ( 82 ); rabbit anti-y + LAT1 ( 83 ); 4F2hc (CD98, M-20, Santa Cruz Biotechnology, Heidelberg, Germany) goat anti-Alix (N-20, sc-49267, Santa Cruz Biotechnology, Heidelberg, Germany); goat anti-Vps35 and goat anti-galectin-3 (Bio-Techne, Abingdon, UK); mouse anti-SARS-CoV-2 Nucleoprotein Antibody (ProSci, 35–579); sheep anti-N-Cadherin (R&D Systems, AF6426); rabbit anti-Megalin (Sigma, SAB1305050); mouse anti-SARS-CoV-2 spike (GeneTex, GTX632604); rabbit anti-cleaved caspase-3 (Cell Signaling, 9661).

Techniques: Two Tailed Test, Western Blot, Immunohistochemical staining, Staining, Labeling

Journal: Science signaling

Article Title: VPS34-dependent control of apical membrane function of proximal tubule cells and nutrient recovery by the kidney

doi: 10.1126/scisignal.abo7940

Figure Lengend Snippet: Transport proteins with reduced phosphorylation.

Article Snippet: The following antibodies were used: rabbit anti-Vps34 (Immobilon); guinea pig anti-megalin ( 81 ); rabbit anti-clathrin (Abcam, Cambridge UK, ab 21679); rabbit anti-EEA1 and rabbit anti-Rab11 both purchased from Invitrogen, rat anti-Lamp2 (sc-19991, Santa Cruz Biotechnology), mouse-anti NKA (MerckMillipore, Germany); rabbit anti-NBC1 (Alomone labs), rabbit and guinea pig anti-SGLT2 (H. Köpsell, University Würzburg), rabbit anti-S6P (5G10), rabbit anti-phospho-S6P (D57.2.2E), both purchased from Cell Signaling Technologies; sheep anti-NDRG1, sheep anti-phospho-NDRG1 (gift of D. Alessi, University of Dundee, UK); rabbit anti-B 0 AT1 ( 82 ); rabbit anti-y + LAT1 ( 83 ); 4F2hc (CD98, M-20, Santa Cruz Biotechnology, Heidelberg, Germany) goat anti-Alix (N-20, sc-49267, Santa Cruz Biotechnology, Heidelberg, Germany); goat anti-Vps35 and goat anti-galectin-3 (Bio-Techne, Abingdon, UK); mouse anti-SARS-CoV-2 Nucleoprotein Antibody (ProSci, 35–579); sheep anti-N-Cadherin (R&D Systems, AF6426); rabbit anti-Megalin (Sigma, SAB1305050); mouse anti-SARS-CoV-2 spike (GeneTex, GTX632604); rabbit anti-cleaved caspase-3 (Cell Signaling, 9661).

Techniques: