Journal: The Journal of Physiology

Article Title: Large‐conductance Ca 2+ ‐activated K + channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium

doi: 10.1113/JP274200

Figure Lengend Snippet: A, representative I SC trace of control epithelia pretreated (20 min) with 500 nm GF109203x, a pan‐specific PKC inhibitor (particularly effective against PKCα and β1), then stimulated with 20 μm UTP (n = 6). Addition of 100 μm DIDS then 50 μm CaCCinh‐AO1, each reduced the I SC. B, histogram summarizing the effects of PKC inhibitors GF109203x and Gö6983 on UTP‐stimulated I SC. C, histogram summarizing the effects of 2 h pretreatment with 100 nm PMA on UTP‐stimulated I SC in control and HC0 epithelia. D, qRT‐PCR analysis of PKC isoform mRNAs (n = 6). Statistical significance (*, P < 0.05) represents differences between relative expression of mRNAs in control and HC0 cells determined by Student's t tests. E, Western blot analysis of PKCα (100%, 94.5%, 94.9%, 94.0%), δ (100%, 106.0%, 99.8%, 94.4%) and ε (100%, 99.7%, 105.5%, 107.9%) in day 8 and day 24 control and HC0 epithelia, respectively. Protein abundance is normalized to β‐actin for each sample and expressed as a percentage of control at day 8. F, qRT‐PCR analysis of KCNMA1 mRNA expression in control and HC) epithelia (n = 6 for each condition). KCNMA1wt was previously shown in Fig. 2 A. STREXi and STREXii refer to two distinct PCR products that contain the STREX exon.

Article Snippet: GAPDH expression was used as the reference gene. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Gene Taqman ID Exon boundary KCNMA1 Hs04334507_m1 1–2 KCNMB1 Hs00188073_m1 3–4 KCNMB2 Hs00175772_m1 3–4 KCNMB3 Hs00205246_m1 2–3 KCNMB4 Hs00205704_m1 2–3 LRRC26 Hs02385555_g1 1–2 P2RY2 Hs04176264_s1 3–3 P2RY4 Hs00267404_s1 1–1 P2RY6 Hs00366312_m1 2–3 PRKCA Hs00925193_m1 14–15 PRKCB Hs00176998_m1 8–9 PRKCD Hs01090047_m1 17–18 PRKCE Hs00942877_m1 13–14 PRKCZ Hs00177051_m1 8–9 KCNMA1 (STREXi) Hs01119492_m1 21–22 KCNMA1 (STREXii) Hs01119490_m1 20–21 Open in a separate window TaqMan probes used in QRT‐PCR assays Immunofluorescence and Western blot analysis NHBE cells grown on Snapwell polyester membranes were fixed in 4% paraformaldehyde for 20 min, as previously described (Zaidman et al . 2016 ).

Techniques: Control, Quantitative RT-PCR, Expressing, Western Blot, Quantitative Proteomics

Journal: The Journal of Physiology

Article Title: Large‐conductance Ca 2+ ‐activated K + channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium

doi: 10.1113/JP274200

Figure Lengend Snippet: TaqMan probes used in QRT‐PCR assays

Article Snippet: GAPDH expression was used as the reference gene. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Gene Taqman ID Exon boundary KCNMA1 Hs04334507_m1 1–2 KCNMB1 Hs00188073_m1 3–4 KCNMB2 Hs00175772_m1 3–4 KCNMB3 Hs00205246_m1 2–3 KCNMB4 Hs00205704_m1 2–3 LRRC26 Hs02385555_g1 1–2 P2RY2 Hs04176264_s1 3–3 P2RY4 Hs00267404_s1 1–1 P2RY6 Hs00366312_m1 2–3 PRKCA Hs00925193_m1 14–15 PRKCB Hs00176998_m1 8–9 PRKCD Hs01090047_m1 17–18 PRKCE Hs00942877_m1 13–14 PRKCZ Hs00177051_m1 8–9 KCNMA1 (STREXi) Hs01119492_m1 21–22 KCNMA1 (STREXii) Hs01119490_m1 20–21 Open in a separate window TaqMan probes used in QRT‐PCR assays Immunofluorescence and Western blot analysis NHBE cells grown on Snapwell polyester membranes were fixed in 4% paraformaldehyde for 20 min, as previously described (Zaidman et al . 2016 ).

Techniques:

Journal: The Journal of Physiology

Article Title: Large‐conductance Ca 2+ ‐activated K + channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium

doi: 10.1113/JP274200

Figure Lengend Snippet: ATP/UTP stimulate Gq‐coupled purinergic receptors expressed on the apical membrane of surface airway epithelial cells leading to increased [Ca2+]i and activation of PKC. PKC‐dependent phosphorylation of BK permits channel activation after stimulation with ATP/UTP, which also activates Ca2+‐activated chloride channels (ANO1/TMEM16A). In control epithelia, HC suppresses transcription of KCNMA1 that contain the STREX exon. In HC0 epithelia, KCNMA1STREX mRNAs are the predominant transcript encoding BKα. The STREX exon makes BKα unresponsive to purinergic receptor activation.

Article Snippet: GAPDH expression was used as the reference gene. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Gene Taqman ID Exon boundary KCNMA1 Hs04334507_m1 1–2 KCNMB1 Hs00188073_m1 3–4 KCNMB2 Hs00175772_m1 3–4 KCNMB3 Hs00205246_m1 2–3 KCNMB4 Hs00205704_m1 2–3 LRRC26 Hs02385555_g1 1–2 P2RY2 Hs04176264_s1 3–3 P2RY4 Hs00267404_s1 1–1 P2RY6 Hs00366312_m1 2–3 PRKCA Hs00925193_m1 14–15 PRKCB Hs00176998_m1 8–9 PRKCD Hs01090047_m1 17–18 PRKCE Hs00942877_m1 13–14 PRKCZ Hs00177051_m1 8–9 KCNMA1 (STREXi) Hs01119492_m1 21–22 KCNMA1 (STREXii) Hs01119490_m1 20–21 Open in a separate window TaqMan probes used in QRT‐PCR assays Immunofluorescence and Western blot analysis NHBE cells grown on Snapwell polyester membranes were fixed in 4% paraformaldehyde for 20 min, as previously described (Zaidman et al . 2016 ).

Techniques: Membrane, Activation Assay, Phospho-proteomics, Control