Journal: Open Life Sciences

Article Title: Effects of KCa channels on biological behavior of trophoblasts

doi: 10.1515/biol-2022-0462

Figure Lengend Snippet: Expression of KCa channels in human villus tissues. Immunohistochemical detection of different types of KCa channels (BKCa α, BKCa β1, IKCa, and SKCa) and the negative control in human villus tissues.

Article Snippet: Then, cells were permeabilized with 0.5% TritonX-100 for 5 min before being blocked with 5% BSA for 1 h. The coverslips were incubated with primary antibody against cytokeratin 7 (CK7, 1:50; Abcam, UK) and anti-vimentin (1:50; Abcam), which were used for the confirmation of EVTs, as well as with other primary antibodies against BKCa α (1:50; Alomone Labs), BKCa β1 (1:50; Alomone Labs), IKCa (1:50; Alomone Labs), and SKCa (1:50; Alomone Labs) at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Negative Control

Journal: Open Life Sciences

Article Title: Effects of KCa channels on biological behavior of trophoblasts

doi: 10.1515/biol-2022-0462

Figure Lengend Snippet: Localization and expression of KCa channels in HTR-8/SVneo cells. BKCa α and β1 subunits, IKCa channels, SKCa channels, and the control in HTR-8/SVneo cells were observed under the fluorescence microscope. Cells were stained with DAPI to visualize nucleus (blue) and immunolabeled with Anti-KCa antibodies (green), respectively. Arrow indicates the positive staining.

Article Snippet: Then, cells were permeabilized with 0.5% TritonX-100 for 5 min before being blocked with 5% BSA for 1 h. The coverslips were incubated with primary antibody against cytokeratin 7 (CK7, 1:50; Abcam, UK) and anti-vimentin (1:50; Abcam), which were used for the confirmation of EVTs, as well as with other primary antibodies against BKCa α (1:50; Alomone Labs), BKCa β1 (1:50; Alomone Labs), IKCa (1:50; Alomone Labs), and SKCa (1:50; Alomone Labs) at 4°C overnight.

Techniques: Expressing, Control, Fluorescence, Microscopy, Staining, Immunolabeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells

doi: 10.1073/pnas.1703081114

Figure Lengend Snippet: Anti-LRRC26 antibody pulls down SLO1 protein in parotid, lacrimal gland, and colon. (A) Confirmation of LRRC26 association with SLO1 protein in mouse parotid. (A1) Total membrane proteins from parotid wt, Slo1−/−, and Lrrc26−/− mice were blotted with anti-SLO1 Ab (L6/60, Antibodies, Inc.), identifying the SLO1 protein in wt and Lrrc26−/− mice, but not Slo1−/− mice. (A2) Proteins immunoprecipitated by the ProSci LRRC26 Ab were Western blotted, showing that LRRC26 is present in both wt and Slo1−/− mice, but not in the LRRC26 KO mice. (A3) Following immunoprecipitation of parotid membrane proteins with the LRRC26 Ab, SLO1 protein is identified in wt immunoprecipitated proteins, but not in Slo1−/− or Lrrc26−/− proteins. (A4) Aliquots of the parotid membrane protein preparations were blotted with a Na/K ATPase1A1 Ab to confirm that similar amounts of proteins were applied in all cases. (B1) Lacrimal gland total membrane proteins were blotted with the anti-SLO1 Ab. (B2) wt and Slo1−/−, but not Lrrc26−/−, lacrimal gland proteins contain LRRC26 protein. (B3) SLO1 protein is found in lacrimal gland membrane proteins immunoprecipitated with the LRRC26 Ab. (B4) Aliquots of lacrimal gland membrane proteins were blotted for ATP1A1. Note the markedly lower amounts of ATP1A1 in lacrimal gland, compared with parotid. (C1–C4) Slo1 protein in colon is also immunoprecipitated with the LRRC26 Ab.

Article Snippet: The coassembly of LRRC26 with SLO1 in parotid gland, lacrimal gland, and colon supports the view that LRRC26 is a BK regulatory subunit in these tissues and predicts that BK gating will be shifted leftward in these cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3. caption a7 Anti-LRRC26 antibody pulls down SLO1 protein in parotid, lacrimal gland, and colon. ( A ) Confirmation of LRRC26 association with SLO1 protein in mouse parotid. ( A1 ) Total membrane proteins from parotid wt , Slo1 −/− , and Lrrc26 −/− mice were blotted with anti-SLO1 Ab (L6/60, Antibodies, Inc.), identifying the SLO1 protein in wt and Lrrc26 −/− mice, but not Slo1 −/− mice. ( A2 ) Proteins immunoprecipitated by the ProSci LRRC26 Ab were Western blotted, showing that LRRC26 is present in both wt and Slo1 −/− mice , but not in the LRRC26 KO mice. ( A3 ) Following immunoprecipitation of parotid membrane proteins with the LRRC26 Ab, SLO1 protein is identified in wt immunoprecipitated proteins, but not in Slo1 −/− or Lrrc26 −/− proteins. ( A4 ) Aliquots of the parotid membrane protein preparations were blotted with a Na/K ATPase1A1 Ab to confirm that similar amounts of proteins were applied in all cases. ( B1 ) Lacrimal gland total membrane proteins were blotted with the anti-SLO1 Ab. ( B2 ) wt and Slo1 −/− , but not Lrrc26 −/− , lacrimal gland proteins contain LRRC26 protein. ( B3 ) SLO1 protein is found in lacrimal gland membrane proteins immunoprecipitated with the LRRC26 Ab. ( B4 ) Aliquots of lacrimal gland membrane proteins were blotted for ATP1A1.

Techniques: Membrane, Immunoprecipitation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells

doi: 10.1073/pnas.1703081114

Figure Lengend Snippet: LRRC26 KO mimics effect of SLO1 KO in reducing K+ efflux in salivary gland secretions. (A) Potassium content was measured from pilocarpine-induced fluid secretion from in vivo parotid glands, for wt (gray) and LRRC26 KO animals. Height of bars shows mean with error bars indicating SEM, whereas circles show individual determinations. Each determination is the average of secretion measured separately from both glands in a single animal, except in one case where only a single gland was obtained. (B) Two bars on the left compare potassium content in submandibular gland saliva from an in vivo measurement, and the two bars on the right show ex vivo potassium content of submandibular salivary secretion. For all comparisons between wt and Lrrc26−/− glands in both A and B, P < 0.001 for the t test.

Article Snippet: The coassembly of LRRC26 with SLO1 in parotid gland, lacrimal gland, and colon supports the view that LRRC26 is a BK regulatory subunit in these tissues and predicts that BK gating will be shifted leftward in these cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3. caption a7 Anti-LRRC26 antibody pulls down SLO1 protein in parotid, lacrimal gland, and colon. ( A ) Confirmation of LRRC26 association with SLO1 protein in mouse parotid. ( A1 ) Total membrane proteins from parotid wt , Slo1 −/− , and Lrrc26 −/− mice were blotted with anti-SLO1 Ab (L6/60, Antibodies, Inc.), identifying the SLO1 protein in wt and Lrrc26 −/− mice, but not Slo1 −/− mice. ( A2 ) Proteins immunoprecipitated by the ProSci LRRC26 Ab were Western blotted, showing that LRRC26 is present in both wt and Slo1 −/− mice , but not in the LRRC26 KO mice. ( A3 ) Following immunoprecipitation of parotid membrane proteins with the LRRC26 Ab, SLO1 protein is identified in wt immunoprecipitated proteins, but not in Slo1 −/− or Lrrc26 −/− proteins. ( A4 ) Aliquots of the parotid membrane protein preparations were blotted with a Na/K ATPase1A1 Ab to confirm that similar amounts of proteins were applied in all cases. ( B1 ) Lacrimal gland total membrane proteins were blotted with the anti-SLO1 Ab. ( B2 ) wt and Slo1 −/− , but not Lrrc26 −/− , lacrimal gland proteins contain LRRC26 protein. ( B3 ) SLO1 protein is found in lacrimal gland membrane proteins immunoprecipitated with the LRRC26 Ab. ( B4 ) Aliquots of lacrimal gland membrane proteins were blotted for ATP1A1.

Techniques: In Vivo, Ex Vivo