rabbit polyclonal nbce1 antibody  (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: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: Primer sequences for reverse transcription quantitative polymerase chain reaction.

Article Snippet: Paraffin sections (4 μm) from 4 TMA were stained with rabbit polyclonal antibody against SLC4A4 (Atlas Antibodies, Sweden, catalogue #HPA035628) at a 1:400 dilution.

Techniques: Reverse Transcription

Journal: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: Upregulation of SLC4A4 in prostate cancer. ( A , B ) SLC4A4 transcript is highly expressed in several cancers, including prostate adenocarcinoma (PRAD), based on the TCGA study cohort. ( C , D ) A subset of PRAD showed higher SLC4A4 expression in Gleason score 7, 8, and 9 tumors and this was associated with the overall survival of patients. ( E , F ) Immunohistochemical (IHC) analysis of primary prostate tumor samples showed higher SLC4A4 protein expression compared to non-tumor tissues. ( G , H ) Compared to RWPE-1 normal prostate epithelial cells, the SLC4A4 mRNA level was higher in DU145 androgen receptor (AR)-negative prostate cancer (PCa) cells. In AR-positive PCa cells, SLC4A4 mRNA level was elevated in C4-2, followed by VCAP (* P < 0.05). SLC4A4 overexpression was also detected by Western blotting, with PC3 cells having higher SLC4A4 protein level in AR-negative PCa cells. In AR-positive PCa cells, both C4-2 and LNCAP showed higher SLC4A4 protein expression. The results are presented as means ± standard error of three independent experiments.

Article Snippet: Paraffin sections (4 μm) from 4 TMA were stained with rabbit polyclonal antibody against SLC4A4 (Atlas Antibodies, Sweden, catalogue #HPA035628) at a 1:400 dilution.

Techniques: Expressing, Immunohistochemical staining, Over Expression, Western Blot

Journal: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: SLC4A4 knockdown suppresses growth, viability and colony formation of prostate cancer (PCa) cells ( A , B ) Western blot analysis and quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed the knockdown of SLC4A4 in PCa cells ( C ) Knockdown of SLC4A4 reduced the growth and viability of PCa cells compared to those transduced with control shRNA (shCON) ( D ) SLC4A4 knockdown reduced colony formation of PCa cells. Cells were previously transfected with control shRNA and SLC4A4 shRNA, seeded in 10 cm Petri dish, and allowed to grow for 2 weeks. The colonies were fixed with 4% paraformaldehyde, stained with crystal violet and imaged using a gel doc. The number of colonies in each sample was counted using Image J software. Graphic representation of the number of colonies showed a significant decrease in the SLC4A4 knockdown group compared to the control (** p < 0.05). The results are presented as means ± standard error of three independent experiments.

Article Snippet: Paraffin sections (4 μm) from 4 TMA were stained with rabbit polyclonal antibody against SLC4A4 (Atlas Antibodies, Sweden, catalogue #HPA035628) at a 1:400 dilution.

Techniques: Knockdown, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Transduction, Control, shRNA, Transfection, Staining, Software

Journal: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: Knockdown of SLC4A4 inhibited PCa invasion and migration. ( A , B ) PCa cells were transfected with control shRNA or SLC4A4 shRNA and subjected to matrigel invasion and migration assays. Cells were seeded into the upper chambers, and after 24 h, cells were examined under a light microscope at 20 × magnification. The results are shown as the mean number of invasive and migrated cells, which were obtained by counting cells in five randomly selected fields. The results are presented as means ± standard error of three independent experiments, with a significant decease observed in the SLC4A4 knockdown group compared to the control (** p < 0.05).

Article Snippet: Paraffin sections (4 μm) from 4 TMA were stained with rabbit polyclonal antibody against SLC4A4 (Atlas Antibodies, Sweden, catalogue #HPA035628) at a 1:400 dilution.

Techniques: Knockdown, Migration, Transfection, Control, shRNA, Light Microscopy

Journal: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: SLC4A4 knockdown restores the activity of GSK-3β and enhances phosphorylation of β-catenin in Prostate Cancer cells ( A ) A significant decrease in the total amount of β-catenin was found in shSLC4A4 prostate cancer cells compared to shCON. Meanwhile, the level of GSK-3β was significantly increased due to its reduced phosphorylation on residues Ser21/9 which led to increase phosphorylation (S33/37) of β-catenin ( B ) Co-immunoprecipitation (Co-IP) experiments showed that endogenous SLC4A4 inhibited the expression of GSK-3β. However, when prostate cancer cells were transfected with the shSLC4A4 plasmid, there was hypophosphorylation of GSK-3β at Ser21/9, which activated the total GSK-3β to degrade endogenous β-catenin ( C ) qPCR data confirmed the degradation of endogenous β-catenin in shSLC4A4 prostate cancer cells.

Article Snippet: Paraffin sections (4 μm) from 4 TMA were stained with rabbit polyclonal antibody against SLC4A4 (Atlas Antibodies, Sweden, catalogue #HPA035628) at a 1:400 dilution.

Techniques: Knockdown, Activity Assay, Phospho-proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: SLC4A4 knockdown induces cell cycle arrest in PCa cells by increasing hypophosphorylation of retinoblastoma protein (RB1) ( A ) SLC4A4 knockdown decreased total RB expression and induced hypophosphorylation at serine 608 (S608) of RB. ( B ) Co-immunoprecipitation (Co-IP) results showed that SLC4A4 KD stable cells had diminished total RB interaction with the SLC4A4 protein ( C ) Cell cycle distribution analysis showed the percentage of PCa cells in G0/G1, S and G2/M. SLC4A4 knockdown induced G0/G1 cell cycle arrest in PCa cells. The results are presented as means ± standard error of three independent experiments.

Article Snippet: Paraffin sections (4 μm) from 4 TMA were stained with rabbit polyclonal antibody against SLC4A4 (Atlas Antibodies, Sweden, catalogue #HPA035628) at a 1:400 dilution.

Techniques: Knockdown, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: SLC4A4 knockdown aberrantly regulates different gene profiles in PCa cells ( A ) The intensities of protein bands detected in western blotting were measured using imageJ (NIH, Bethesda, MD, USA), OriginPro 2021 software (OriginLab Corporation, Northampton, MA, USA) was used to perform hierarchical clustering and heatmap analysis of protein expression/activation in PCa cells either control shRNA (shCON) or SLC4A4 shRNA (shSLC4A4). This analysis identified the coupling relationships that reveal the regulatory pathways driving cell proliferation. ( B ) A heatmap showing top 40 up‐ and downregulated genes after SLC4A4 knockdown in DU145 cells. ( C ) Volcano plot showing distribution of 1157 differentially expressed genes upon SLC4A4 knockdown in DU145 cells. Log2 fold change is represented in x‐axis, while y axis represents –log10 P value. Upregulated and downregulated genes are marked with dots ( D ) qPCR validation of the RNA Sequencing data in DU-145 cells ( E ) Gene Ontology (GO) biological process enrichment analysis of the genes differentially expressed upon SLC4A4 knockdown in Prostate Cancer (PCa) cells. ( F ) Members of the SLC4 transporter family that were altered after SLC4A4 knockdown in PCa cells.

Article Snippet: Paraffin sections (4 μm) from 4 TMA were stained with rabbit polyclonal antibody against SLC4A4 (Atlas Antibodies, Sweden, catalogue #HPA035628) at a 1:400 dilution.

Techniques: Knockdown, Western Blot, Software, Expressing, Activation Assay, Control, shRNA, Biomarker Discovery, RNA Sequencing

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 1. SLC4A4 was downregulated in LUAD tissues and cell lines. (A) The expression of SLC4A4 in normal and tumor tissues was detected by qRT-PCR and analyzed for relative quantification; (B) The expression of SLC4A4 in normal and tumor tissues was detected by WB and analyzed for relative quantification; (C) IHC was used to detect and quantify the expression of SLC4A4 in normal and tumor tissues; (D) The expression of SLC4A4 in A549 cell line was detected by qRT-PCR and WB and the qRT-PCR results were analyzed for relative quantification; (E) The expression of SLC4A4 in H1299 cell line was detected by qRT-PCR and WB and the qRT-PCR results were analyzed for relative quantification. ** means P<0.01, and *** means P<0.001.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 2. SLC4A4 inhibited the migration and invasive ability of LUAD cell lines. (A) The efficiency of overexpression of SLC4A4 in A549 and H1299 cell lines was detected and quantified; (B-C) CCK8 detects changes in cell viability before and after overexpression of SLC4A4 in A549 (B) and H1299 (C) cell lines; (D-G) Transwell assay for changes in cell invasion and migration ability before and after overexpression of SLC4A4 in A549 (D-E) and H1299 (F-G) cell lines; (H) Changes in apoptosis rate before and after SLC4A4 overexpression in A549 and H1299 cell lines.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Migration, Over Expression, Transwell Assay

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 3. SLC4A4 inhibited EMT in LUAD cell lines and inhibits angiogenesis. (A-D) The expression of MMP9 (A), E-CAD (B), N-CAD (C), and VIM (D) in A549 and H1299 cell lines before and after overexpression of SLC4A4 was detected by qRT-PCR and analyzed for relative quantification; (E) Determination of proliferative capacity of HUVECs after SLC4A4 overexpression; (F) Live-dead cells were assayed for the level of apoptosis in HUVECs after stimulation of HUVECs with conditioned medium before and after overexpression of SLC4A4 in LUAD cell lines.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Quantitative Proteomics

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 4. Relationship between SLC4A4 expression and immune checkpoint genes in public databases. (A) Heat map of checkpoint gene correlation analysis with SLC4A4 gene in pan-cancer; (B) Correlation of SLC4A4 with PD-1 gene in TCGA-LUAD dataset; (C) Correlation of SLC4A4 with PD-1 gene in GSE50081 dataset; (D) Bubble plot of SLC4A4 correlation with other checkpoint genes in TCGA-LUAD dataset.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Expressing

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 5. Correlation between the expression of SLC4A4 and immune characteristics. (A) The heat map of SLC4A4 expression level and immune cell correlation distribution in pan-cancers; (B) Correlation of SLC4A4 gene with ESTIMATE scores, immune scores and stromal scores in pan-cancers. * represented P<0.05, ** represented P<0.01, and *** represented P<0.001.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Expressing

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 6. The expression profile, immune and stromal scores are related to the biological functions of DEGs. (A-C) Volcano plots showing the differently expressed genes of SLC4A4 expression, Immune and Stromal score (high vs. low). (D, E) Intersection between SLC4A4 expression and immune/stromal scores and DEGs.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Expressing

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 7. Construction of a risk model associated with SLC4A4 and prediction of immunotherapy response. (A) Univariate Cox regression analysis of 687 DEGs; (B) Univariate Cox regression analysis based on 319 genes, with the ordinate indicating the coefficients of the variables and the abscissa indicating the logarithm of the optimal parameter (lambda); (C) Reaching lambda by a combination of at least 23 genes; (D) Distribution and expression of patient survival status for 10 gene construct risk scores in the TCGA-LUAD dataset; (E) Correlation analysis between the expression of SLC4A4 and risk score; (F) Comparison between immune- responsive and non-immune-responsive groups with risk score and (G) SLC4A4 expression；(H) Correlation analysis between risk score and TIDE; (I) Correlation analysis between SLC4A4A expression and TIDE. **** represented P<0.0001, and ** represented P < 0.01.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Expressing, Construct, Comparison

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 8. SLC4A4 promoted the infiltration of immune cells in tumor tissues. (A) The size of tumor volume before and after overexpression of SLC4A4 in the A549 and H1299 cell lines was quantified; (B) Strategies for detecting immune cells by flow cytometry; (C-G) The number of infiltrating neutrophils (C), monocytes (D), CD4+ T cells (E), CD8+ T cells (F), and B cells in tumor tissues was quantified.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Over Expression, Flow Cytometry

Journal: International immunopharmacology

Article Title: SLC4A4 as a novel biomarker involved in immune system response and lung adenocarcinoma progression.

doi: 10.1016/j.intimp.2024.112756

Figure Lengend Snippet: Fig. 9. Functional assays for CD8+ T cells. (A) Ki67 expression in CD8+ T cell populations before and after SLC4A4 overexpression and quantified; (B-C) Differences in the expression levels of IFN-γ (B) and GzmB (C) after SLC4A4 overexpression; (D) Expression levels of PD-1 in CD8+ T cells before and after SLC4A4 overexpression.

Article Snippet: Deparaffinized and dehydrated sections were further incubated with polyclonal rabbit anti-SLC4A4 antibodies (1:500, Proteintech, 11885–1- AP) at 4 ◦C overnight and then with secondary antibodies (1:1000, Proteintech, SA00001-2) at room temperature for 2 h. Signal detection was achieved using enhanced DAB staining kit (Proteintech, China).

Techniques: Functional Assay, Expressing, Over Expression

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: