Journal: American Journal of Physiology - Renal Physiology
Article Title: H + -ATPase B1 subunit localizes to thick ascending limb and distal convoluted tubule of rodent and human kidney
doi: 10.1152/ajprenal.00539.2017
Figure Lengend Snippet: H+-ATPase B1 subunit is expressed in apical membrane domains along the early portion of the mouse distal nephron. A and B: immunostaining for H+-ATPase B1 subunit (ATP6V1B1) in mouse kidney cross section with the ATP6V1B1H7659 antibody in the cortex (A) and the medulla (B). In the cortex, expression was evident in the intercalated cells (ICs) as well as in early distal tubular segments. In the outer medulla, both thick ascending limb (TAL) segments and ICs in collecting ducts were positive for ATP6V1B1 with the ATP6V1B1H7659 antibody. Toward the inner medullary portion of the kidney, staining was visible only in the ICs of the collecting duct system. Gray stippled lines demarcate the zones of the medulla. C: representative confocal images showing detection of the ATP6V1B1 with the TAL marker SLC12A1 (green) and the ATP6V1B1H7659 antibody (red). Image from the cortical TAL. Staining is visible in ICs as shown by an arrowhead and in TAL as shown by an asterisk. D: confocal images showing localization of ATP6V1B1 with the ATP6V1B1H7659 antibody (red) with the distal convoluted tubule (DCT) marker, SLC12A3 (green). Again, an arrowhead indicates 1 IC, and a DCT tubule is shown by an asterisk. E: immunofluorescent staining with antibodies directed against the H+-ATPase B1 subunit with the ATP6V1B1H7659 antibody (red) with plasma membrane Ca2+-ATPase 4 (PMCA4; green). Staining is present across the cortical TAL, the DCT (DCT1 and DCT2), in addition to the ICs of the collecting system (arrowhead). F: representative confocal images showing detection of ATP6V1B1 with the ATP6V1B1H7659 antibody (red) with AQP2 (green). ICs are indicated by arrowheads, and early distal nephron tubules are marked with an asterisk. G: confocal images showing immunofluorescence labeling of enhanced green fluorescent protein (eGFP; green) with SLC12A3 in transgenic mice expressing eGFP after a 6.5-kb fragment of the ATP6V1B1 promoter. eGFP expression is visible in the collecting system [including both connecting tubule (CNT) cells and ICs, the latter indicated by arrowhead] but absent from DCT defined by SLC12A3 expression as indicated by an asterisk. H: representative confocal images showing detection of eGFP (green) and ATP6V1B1 (red) with the ATP6V1B1H7659 antibody in ATP6V1B1-eGFP mice. ICs are indicated by arrowheads, and early distal tubular segments are indicated by an asterisk. Weak immunoreactivity for ATP6V1B1 is visible in apical domains of CNT cells that express eGFP (indicated by arrowheads).
Article Snippet: Furthermore, the following antibodies were used to determine localization of other acid-base transporters and specific markers for individual nephron segments throughout the renal tubule: 1 ) goat polyclonal antibodies against Aquaporin 2 (AQP2; C-17; Santa Cruz Biotechnology); 2 ) rabbit polyclonal antibodies against NKCC2 (SLC12A1, HPA014967; Sigma-Aldrich); 3 ) rabbit polyclonal antibodies against NCC (SLC12A3 HPA028748; Sigma-Aldrich); 4 ) mouse monoclonal antibody against plasma membrane Ca 2+ -ATPase 4 (PMCA4) (JA9, ab2783; Abcam, Cambridge, UK); 5 ) goat polyclonal antibodies against eGFP (Abcam); 6 ) rabbit polyclonal antibodies against ATP6V1E1 (PA5-29899, Thermo Fisher Scientific, Slangerup, Denmark); 7 ) rabbit polyclonal antibodies against ATP6V1B2 (HPA008147; Sigma-Aldrich); 8 ) rabbit polyclonal antibodies against ATP6V1G1 (PA00135A0Rb; Cusabio Technologies, Houston TX); 9 ) mouse monoclonal antibodies directed against the human thiazide-sensitive NaCl cotransporter encoded by the SLC12A3 gene generated as described in detail previously ( 39 ).
Techniques: Immunostaining, Expressing, Staining, Marker, Immunofluorescence, Labeling, Transgenic Assay
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![SP1 inhibits expression of the 1500 bp cldn14 promoter reporter construct. (A) Luciferase activity from the Cldn14 reporter construct after transfection with the transcription factor SP1. SP1 cotransfection significantly decreased luciferase activity of cells cotransfected with Empty Vector (EV) or the CaSR under low and high Ca 2+ concentrations. Ordinary one‐way ANOVA was used to determine statistical difference (*** /### p < .001; ## p < .01; # p < .05). (B) Immunoblots demonstrating co‐expression of the CaSR with SP1. (C) Immunohistochemical staining of a murine renal section for SP1 (brown) and <t>NKCC2</t> (blue). (D) high magnification image of thick ascending limb with nuclear localization of SP1. (E) Representative immunoblots of myc tagged SP1 run on regular or FeCl 3 ‐loaded gels. (F) Relative phospho‐SP1 protein abundance decreased when cells were incubated in with high [Ca 2+ ] and expressed the CaSR. Friedman test with Dunn's multiple comparisons test were used to determine statistical differences (* p < .05)](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7942/pmc09297942/pmc09297942__FSB2-35-0-g004.jpg)
![H+-ATPase B1 subunit is expressed in apical membrane domains along the early portion of the mouse distal nephron. A and B: immunostaining for H+-ATPase B1 subunit (ATP6V1B1) in mouse kidney cross section with the ATP6V1B1H7659 antibody in the cortex (A) and the medulla (B). In the cortex, expression was evident in the intercalated cells (ICs) as well as in early distal tubular segments. In the outer medulla, both thick ascending limb (TAL) segments and ICs in collecting ducts were positive for ATP6V1B1 with the ATP6V1B1H7659 antibody. Toward the inner medullary portion of the kidney, staining was visible only in the ICs of the collecting duct system. Gray stippled lines demarcate the zones of the medulla. C: representative confocal images showing detection of the ATP6V1B1 with the TAL marker <t>SLC12A1</t> (green) and the ATP6V1B1H7659 antibody (red). Image from the cortical TAL. Staining is visible in ICs as shown by an arrowhead and in TAL as shown by an asterisk. D: confocal images showing localization of ATP6V1B1 with the ATP6V1B1H7659 antibody (red) with the distal convoluted tubule (DCT) marker, SLC12A3 (green). Again, an arrowhead indicates 1 IC, and a DCT tubule is shown by an asterisk. E: immunofluorescent staining with antibodies directed against the H+-ATPase B1 subunit with the ATP6V1B1H7659 antibody (red) with plasma membrane Ca2+-ATPase 4 (PMCA4; green). Staining is present across the cortical TAL, the DCT (DCT1 and DCT2), in addition to the ICs of the collecting system (arrowhead). F: representative confocal images showing detection of ATP6V1B1 with the ATP6V1B1H7659 antibody (red) with AQP2 (green). ICs are indicated by arrowheads, and early distal nephron tubules are marked with an asterisk. G: confocal images showing immunofluorescence labeling of enhanced green fluorescent protein (eGFP; green) with SLC12A3 in transgenic mice expressing eGFP after a 6.5-kb fragment of the ATP6V1B1 promoter. eGFP expression is visible in the collecting system [including both connecting tubule (CNT) cells and ICs, the latter indicated by arrowhead] but absent from DCT defined by SLC12A3 expression as indicated by an asterisk. H: representative confocal images showing detection of eGFP (green) and ATP6V1B1 (red) with the ATP6V1B1H7659 antibody in ATP6V1B1-eGFP mice. ICs are indicated by arrowheads, and early distal tubular segments are indicated by an asterisk. Weak immunoreactivity for ATP6V1B1 is visible in apical domains of CNT cells that express eGFP (indicated by arrowheads).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2570/pmc06172570/pmc06172570__zh20091885910002.jpg)
