Journal: International Journal of Molecular Sciences

Article Title: The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

doi: 10.3390/ijms22157790

Figure Lengend Snippet: VPA increases cell surface expression of TrkC. ( A , B ) SH-SY5Y and BE(2)-C cells were incubated for 24 h with either vehicle or 1 mM VPA. Cells were then treated with the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido) hexanoate and solubilized proteins were isolated by precipitation with streptavidin-conjugated agarose beads. The total extract (cell lysate) and precipitated proteins (surface protein) were analyzed for TrkC by Western blot. The bar graphs show the changes in cell surface TrkC levels normalized to pan-cadherin (pan-cadh) levels, used as plasma membrane marker. Values are the mean ± SD of four independent experiments. ( C ) SH-SY5Y cells grown onto glass coverslips and treated for 24 h with either vehicle or 1 mM VPA were analyzed for TrkC expression by immunofluorescence without permeabilization and with an antibody recognizing an extracellular domain of the receptor (green color). Nuclei were stained in blue with 4′,6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bar, 25 µm. Values reported in the scatterplot are the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. control (vehicle) by Student’s t test. ( D , E ) SH-SY5Y cells were incubated in serum-free medium with either vehicle or 1 mM VPA for 24 h and then exposed for 5 min to either vehicle or 1 nM NT-3. Cell lysates were analyzed for the expression of phospho-Thr308-Akt (pAkt) and total Akt ( D ) and phospho-ERK1/2 (pERK1/2) and total ERK1/2 ( E ). Values are the mean ± SD of four independent experiments. * p < 0.05 vs. control (vehicle + vehicle); ## p < 0.01, ### p < 0.001 vs. VPA + vehicle by one-way analysis of variance (ANOVA) followed by Tukey’s test.

Article Snippet: Membranes were blocked with 5% low-fat dry milk, washed and incubated overnight at 4 °C with one of the following primary antibodies: TrkC (cat. no. 3376, Cell Signaling Technology) (1:1000), TrkC-T1 (cat no. 600-401-993, Rockland, Limerick, PA, USA) (1:1000), p75NTR (cat no. 8238, Cell Signaling Technology) (1:1000), Jun N-terminal kinase (JNK) (sc-571, Santa Cruz Biotechnology) (1:2000), phospho-JNK (Thr183/Tyr185) (cat. no. 9912, Cell Signaling Technology) (1:1000), phospho-c-Jun (Ser73) (cat. no. 3270, Cell Signaling Technology) (1:1000), c-Jun (cat. no. 9165, Cell Signaling Technology) (1:1000), phospho-Akt (Thr308) (cat. no. 2965, Cell Signaling Technology) (1:5000), Akt1/2/3 (sc-8312, Santa Cruz Biotechnology) (1:1000), extracellular signal-regulated kinases 1 and 2 (ERK1/2) (cat no. 9102, Cell Signaling Technology), phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (cat no. RA15002, Neuromics, Nothfield, MN, USA) (1:5000), phospho-MEK1/2 (Ser217/221) (cat no. 9154, Cell Signaling Technology) (1:1000), MEK1/2 (sc-81504, Santa Cruz Biotechnology) (1:1000), phospho-Elk-1 (Ser383) (cat. no. 9181, Cell Signaling Technology) (1:1000), Elk-1 (sc-365876, Santa Cruz Biotechnology) (1:1000), RUNX3/AML2 (RUNX3) (cat no. 9647, Cell Signaling Technology) (1:1000), RUNX1/AML1 (AML1) (cat. no. 4336, Cell Signaling Technology) (1:1000), phospho-AML1 (Ser249) (cat. no. 4327, Cell Signaling Technology) (1:1000), AP-2α (cat. 3215, Cell Signaling Technology) (1:1000), Egr1 (cat. no. 4154, Cell Signaling Technology) (1:1000), Sp1 (cat. no. 9389, Cell Signaling Technology) (1:1000), p53 (sc-6243, Santa Cruz Biotechnology) (1:500), HDAC1 (sc-81598, Santa Cruz Biotechnology) (1:2000), poly(ADP-ribose)polymerase (PARP) (cat. no. 9542, Cell Signaling Technology) (1:1000), pan-cadherin (cat no. 4073, Cell Signaling Technology) (1:2000); acetylated tubulin (cat. no. T7451, Sigma-Aldrich) (1:1000), α-tubulin (sc-5286, Santa Cruz Biotechnology) (1:1000), histone H3 (acetyl Lys9, Lys14) (cat no. GTX122648, GeneTex Inc., Irvine, CA, USA) (1:1000), histone H3 (cat. no. GTX 122148, GeneTex Inc.) (1:2000), actin (cat. no. A2066, Sigma-Aldrich) (1:2000), glyceradheyde-3-phosphate-dehydrogenase (GAPDH) (cat no. 247002, Synaptic Systems GmbH, Gottingen, Germany) (1:10,000).

Techniques: Expressing, Incubation, Isolation, Western Blot, Marker, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

doi: 10.3390/ijms22157790

Figure Lengend Snippet: Participation of ERK1/2 and JNK in VPA-induced TrkC upregulation. ( A – F ) SH-SY5Y cells were incubated in serum-free medium with 1 mM VPA for the indicated periods of time. Zero time samples were treated with vehicle and used as control. Cell lysates were analyzed for phospho-MEK1/2 (pMEK1/2), MEK1/2 ( A ), phospho-ERK1/2, ERK1/2 ( B ), phospho-Ser383-Elk-1 (pElk-1), Elk-1 ( C ), phospho-JNK (pJNK), JNK ( D ), phospho-Ser73-cJun (p-cJun), cJun, GAPDH ( E ), TrkC and actin ( F ). Values are the mean ± SD of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. ( G – I ) SH-SY5Y cells were preincubated for 1 h with either vehicle, 25 µM PD 98,059 (PD) ( G ), 10 µM TCS JNK6o (TCS) ( H ), or 1 µM BIRB 0796 (BIRB) ( I ) and then exposed to either vehicle or 1 mM VPA for 24 h. Cell lysates were analyzed for TrkC expression. Values are the mean ± SD of four experiments. *** p < 0.001 vs. control (vehicle + vehicle); ### p < 0.001 vs. vehicle + VPA by ANOVA followed by Tukey’s test.

Article Snippet: Membranes were blocked with 5% low-fat dry milk, washed and incubated overnight at 4 °C with one of the following primary antibodies: TrkC (cat. no. 3376, Cell Signaling Technology) (1:1000), TrkC-T1 (cat no. 600-401-993, Rockland, Limerick, PA, USA) (1:1000), p75NTR (cat no. 8238, Cell Signaling Technology) (1:1000), Jun N-terminal kinase (JNK) (sc-571, Santa Cruz Biotechnology) (1:2000), phospho-JNK (Thr183/Tyr185) (cat. no. 9912, Cell Signaling Technology) (1:1000), phospho-c-Jun (Ser73) (cat. no. 3270, Cell Signaling Technology) (1:1000), c-Jun (cat. no. 9165, Cell Signaling Technology) (1:1000), phospho-Akt (Thr308) (cat. no. 2965, Cell Signaling Technology) (1:5000), Akt1/2/3 (sc-8312, Santa Cruz Biotechnology) (1:1000), extracellular signal-regulated kinases 1 and 2 (ERK1/2) (cat no. 9102, Cell Signaling Technology), phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (cat no. RA15002, Neuromics, Nothfield, MN, USA) (1:5000), phospho-MEK1/2 (Ser217/221) (cat no. 9154, Cell Signaling Technology) (1:1000), MEK1/2 (sc-81504, Santa Cruz Biotechnology) (1:1000), phospho-Elk-1 (Ser383) (cat. no. 9181, Cell Signaling Technology) (1:1000), Elk-1 (sc-365876, Santa Cruz Biotechnology) (1:1000), RUNX3/AML2 (RUNX3) (cat no. 9647, Cell Signaling Technology) (1:1000), RUNX1/AML1 (AML1) (cat. no. 4336, Cell Signaling Technology) (1:1000), phospho-AML1 (Ser249) (cat. no. 4327, Cell Signaling Technology) (1:1000), AP-2α (cat. 3215, Cell Signaling Technology) (1:1000), Egr1 (cat. no. 4154, Cell Signaling Technology) (1:1000), Sp1 (cat. no. 9389, Cell Signaling Technology) (1:1000), p53 (sc-6243, Santa Cruz Biotechnology) (1:500), HDAC1 (sc-81598, Santa Cruz Biotechnology) (1:2000), poly(ADP-ribose)polymerase (PARP) (cat. no. 9542, Cell Signaling Technology) (1:1000), pan-cadherin (cat no. 4073, Cell Signaling Technology) (1:2000); acetylated tubulin (cat. no. T7451, Sigma-Aldrich) (1:1000), α-tubulin (sc-5286, Santa Cruz Biotechnology) (1:1000), histone H3 (acetyl Lys9, Lys14) (cat no. GTX122648, GeneTex Inc., Irvine, CA, USA) (1:1000), histone H3 (cat. no. GTX 122148, GeneTex Inc.) (1:2000), actin (cat. no. A2066, Sigma-Aldrich) (1:2000), glyceradheyde-3-phosphate-dehydrogenase (GAPDH) (cat no. 247002, Synaptic Systems GmbH, Gottingen, Germany) (1:10,000).

Techniques: Incubation, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

doi: 10.3390/ijms22157790

Figure Lengend Snippet: Putative molecular mechanisms mediating the induction of TrkC expression by VPA in human neuroblastoma cells. The diagram illustrates that VPA enhances the expression of the NTRK3 gene encoding TrkC through the following mechanisms: ( a ) epigenetic changes involving RUNX3 derepression as the consequence of HDAC inhibition and EZH2 depletion [ , ]; ( b ) activation of ERK1/2 and JNK signaling pathways. ERK1/2 activation exerts a major stimulatory input on NTRK3 activation by inducing Egr1 expression likely via Elk-1 phosphorylation. The stimulation of JNK appears to provide a minor contribution possibly by participating in Elk-1 phosphorylation.

Article Snippet: Membranes were blocked with 5% low-fat dry milk, washed and incubated overnight at 4 °C with one of the following primary antibodies: TrkC (cat. no. 3376, Cell Signaling Technology) (1:1000), TrkC-T1 (cat no. 600-401-993, Rockland, Limerick, PA, USA) (1:1000), p75NTR (cat no. 8238, Cell Signaling Technology) (1:1000), Jun N-terminal kinase (JNK) (sc-571, Santa Cruz Biotechnology) (1:2000), phospho-JNK (Thr183/Tyr185) (cat. no. 9912, Cell Signaling Technology) (1:1000), phospho-c-Jun (Ser73) (cat. no. 3270, Cell Signaling Technology) (1:1000), c-Jun (cat. no. 9165, Cell Signaling Technology) (1:1000), phospho-Akt (Thr308) (cat. no. 2965, Cell Signaling Technology) (1:5000), Akt1/2/3 (sc-8312, Santa Cruz Biotechnology) (1:1000), extracellular signal-regulated kinases 1 and 2 (ERK1/2) (cat no. 9102, Cell Signaling Technology), phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (cat no. RA15002, Neuromics, Nothfield, MN, USA) (1:5000), phospho-MEK1/2 (Ser217/221) (cat no. 9154, Cell Signaling Technology) (1:1000), MEK1/2 (sc-81504, Santa Cruz Biotechnology) (1:1000), phospho-Elk-1 (Ser383) (cat. no. 9181, Cell Signaling Technology) (1:1000), Elk-1 (sc-365876, Santa Cruz Biotechnology) (1:1000), RUNX3/AML2 (RUNX3) (cat no. 9647, Cell Signaling Technology) (1:1000), RUNX1/AML1 (AML1) (cat. no. 4336, Cell Signaling Technology) (1:1000), phospho-AML1 (Ser249) (cat. no. 4327, Cell Signaling Technology) (1:1000), AP-2α (cat. 3215, Cell Signaling Technology) (1:1000), Egr1 (cat. no. 4154, Cell Signaling Technology) (1:1000), Sp1 (cat. no. 9389, Cell Signaling Technology) (1:1000), p53 (sc-6243, Santa Cruz Biotechnology) (1:500), HDAC1 (sc-81598, Santa Cruz Biotechnology) (1:2000), poly(ADP-ribose)polymerase (PARP) (cat. no. 9542, Cell Signaling Technology) (1:1000), pan-cadherin (cat no. 4073, Cell Signaling Technology) (1:2000); acetylated tubulin (cat. no. T7451, Sigma-Aldrich) (1:1000), α-tubulin (sc-5286, Santa Cruz Biotechnology) (1:1000), histone H3 (acetyl Lys9, Lys14) (cat no. GTX122648, GeneTex Inc., Irvine, CA, USA) (1:1000), histone H3 (cat. no. GTX 122148, GeneTex Inc.) (1:2000), actin (cat. no. A2066, Sigma-Aldrich) (1:2000), glyceradheyde-3-phosphate-dehydrogenase (GAPDH) (cat no. 247002, Synaptic Systems GmbH, Gottingen, Germany) (1:10,000).

Techniques: Expressing, Inhibition, Activation Assay

Journal: Cell reports

Article Title: Tuberculosis exacerbates HIV-1 infection through IL-10/STAT3-dependent tunneling nanotube formation in macrophages

doi: 10.1016/j.celrep.2019.02.091

Figure Lengend Snippet: (A) Vertical scatter plots showing the Median Fluorescence Intensity (MFI) of cell-surface receptors involved in HIV-1 entry (CD4, CCR5, CXCR4) on monocytes differentiated for 3 days under the presence of CmCTR and CmMTB. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. (B) Histogram showing the percentage of HIV-1 fusion with CmCTR- (white) or CmMTB (black)-pre-treated cells, as determined using the Blam-Vpr assay in the presence of entry inhibitor Maraviroc (dashed bars). (C) Left: Representative images of Western Blot analysis illustrating the expression of IFITM1/2/3 and Actin as loading control. Right: Quantification of IFITM1/2/3 expressed as a ratio related to actin of monocytes differentiated for 3 days into macrophages under the presence of CmCTR (white) and CmMTB (black). n = 6 donors. (D) Representative images of Western Blot analysis illustrating the expression of SAMHD1 and its phosphorylated version (pSAMHD1), and Actin as loading control (left). Quantification of SAMHD1 (center) pSAMHD1 (right) expressed as a ratio related to actin of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 11 donors. (E) Representative images of Western Blot analysis illustrating the expression of C/EBP-β (LAP), C/EBP-β (LIP), and Actin as loading control (left). Quantification of C/EBP-β (LAP, center left) and C/EBP-β (LIP, center right) expressed as a ratio related to actin, and LAP expressed as a ratio related to LIP (right), of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 9 donors. LAP is an activator of HIV-1-LTR whereas LIP is a repressor of HIV-1-LTR. (F) Representative images of Western Blot analysis illustrating the expression of CUGBP1 and Actin as loading control (left). Quantification of CUGBP1 expressed as a ratio related to actin (right) of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 9 donors. (G) Quantification of LC3-II expression as a ratio to actin of monocytes differentiated for 3 days under the presence of CmCTR and CmMTB at the indicated time points after 2h treatment with Bafilomycin A1 (BafA1) or DMSO as control, as measured by western blot analysis. Uninfected cells at day 3 of the experiment (left, n = 6 donors), and HIV-infected cells at 1 (day 4, center, n = 4 donors) and 3 (day 6, right, n = 6 donors) days post-infection. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. Data in histograms are represented as mean ± SD. * p≤0.05; ** p≤0.005; *** p≤0.0005; **** p≤0.0001.

Article Snippet: Anti-human phosphoThr592-SAMHD1 , ProSci , Cat# 8005.

Techniques: Fluorescence, Western Blot, Expressing, Infection

Journal: Cell reports

Article Title: Tuberculosis exacerbates HIV-1 infection through IL-10/STAT3-dependent tunneling nanotube formation in macrophages

doi: 10.1016/j.celrep.2019.02.091

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-human phosphoThr592-SAMHD1 , ProSci , Cat# 8005.

Techniques: Purification, Recombinant, Luciferase, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, Software, Imaging

Journal: Neural Regeneration Research

Article Title: Poly-L-ornithine blocks the inhibitory effects of fibronectin on oligodendrocyte differentiation and promotes myelin repair

doi: 10.4103/1673-5374.353493

Figure Lengend Snippet: PLO and FN function through ERK and PI3K/Akt signaling pathways. (A) The diagram shows the integrins (αV, β1, β3, β5) expressed in oligodendrocytes that specifically bind to FN. Data are collected from published literature. (B) Quantitative analysis of the expression levels (normalized by the PDL group) of integrins (αV, β1, β3, β5) during the proliferation and differentiation stages of oligodendrocytes by real-time quantitative polymerase chain reaction. (C) Representative western blots of Akt, P-Akt (Thr308), S6K, P-S6K, Erk1/2, and P-Erk1/2 proteins from OPCs cultured with different coating substances for 2 days in proliferation medium (2 d PM). (D) Quantification of the western blot results (normalized by the PDL group) showed in C. (E) Representative western blots of GSK3β, P-GSK3β, CREB, P-CREB, Akt, P-Akt, Erk, and P-Erk proteins from cells cultured for 1 day in differentiation medium (1d DM). (F) Quantification (normalized by the PDL group) of the results in E. (G) Representative western blots of Akt, P-Akt, Erk, P-Erk, GSK3β, P-GSK3β, CREB, and P-CREB proteins from cells cultured for 3 days in differentiation medium (3d DM). (H) Quantification (normalized by the PDL group) of the results in G. All data are expressed as mean ± SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test). Akt: Protein kinase B; CREB: cyclic adenosine monophosphate response element binding protein; DM: differentiation medium; Erk1/2: extracellular signal-regulated kinases 1 and 2; FN: fibronectin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase 3 beta; P-Akt; phospho-protein kinase B; P-CREB: phospho-cyclic adenosine monophosphate response element binding protein; PDL: poly-D-lysine; P-Erk1/2: phospho-extracellular signal-regulated kinases 1 and 2; P-GSK3β: phospho-glycogen synthase kinase 3 beta; PI3K: phosphoinositide 3-kinase; PLO: poly-L-ornithine; PM: proliferation medium; P-S6k: phospho-ribosomal S6 kinase; S6k: ribosomal S6 kinase.

Article Snippet: The primary antibodies were as follows: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000, rabbit, Proteintech, Cat# 10494-1-AP, RRID: AB_2263076), protein kinase B (PKB, known as Akt; 1:1000, rabbit, Cell Signaling Technology, Cat# 9272, RRID: AB_329827), phospho-protein kinase B (Ser473) (P-PKB, known as P-AKT; 1:1000, rabbit, Cell Signaling Technology, Cat# 9271, RRID: AB_329825), cyclic adenosine monophosphate response element binding protein (CREB; 1:1000, rabbit, Cell Signaling Technology, Cat# 9197, RRID: AB_331277), phospho-cyclic adenosine monophosphate response element binding protein (P-CREB; 1:1000, rabbit, Cell Signaling Technology, Cat# 9198, RRID: AB_2561044), extracellular signal-regulated kinases 1 and 2 (Erk1/2; 1:1000, rabbit, Cell Signaling Technology, Cat# 4695, RRID: AB_390779), phospho-extracellular signal-regulated kinases 1 and 2 (Thr202/Tyr204) (P-Erk1/2; 1:1000, rabbit, Cell Signaling Technology, Cat# 4370, RRID: AB_2315112); glycogen synthase kinase 3 beta (GSK3β; 1:1000, rabbit, Affinity, Cincinnati, OH, USA, Cat# AF5016, RRID: AB_2834935), phospho-glycogen synthase kinase 3 beta (P-GSK3β; 1:1000, rabbit, Affinity, Cat# AF2016, RRID: AB_2834439), ribosomal S6 kinase (S6Κ; 1:1000, rabbit, Affinity, Cat# AF6226, RRID: AB_2835100), phospho-ribosomal S6 kinase (P-S6K; 1:1000, rabbit, Affinity, Cat# AF3228, RRID: AB_2834654), CyclinD1 (1:1000, rabbit, Abcam, Cat# AB134175, RRID: AB_2750906), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP; 1:1000, mouse, Calbiochem, Darmstadt, Germany, Cat# NE1020, RRID: AB_2043400), and myelin basic protein (MBP; 1:1000, mouse, Calbiochem, Cat# NE1018, RRID: AB_2140494).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Binding Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Venetoclax is a potent hepsin inhibitor that reduces the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells

doi: 10.3389/fmolb.2023.1182925

Figure Lengend Snippet: pSTAT3, pAKT and pERK1/2 expression in Caco-2 and Caco-2-HPN cells. Expression of pERK1/2 (A) , pSTAT3 (B) and pAKT (C) determined by electrophoresis and Western blot in lysates of Caco-2 and Caco-2-HPN cells in triplicates. Levels were determined by densitometry and represented as relative protein expression to vinculin. Graphs represent the mean ± standard error of the mean of the triplicates. (D) Electrophoresis and Western blot of pSTAT3, pAKT and pERK1/2 in lysates of Caco-2 and Caco-2-HPN cells in triplicates. Vinculin expression was detected as loading control. pERK1/2 , phospho-extracellular signal-regulated kinases 1 and 2; pSTAT3 , phospho-signal transducer and activator of transcription 3; pAKT , phospho-Protein Kinase B; Caco-2-HPN , Caco-2 cells overexpressing hepsin; Caco-2 , Caco-2 cells with hepsin basal expression; * : p -value < 0.05; HPN , Hepsin; + , overexpression; - , basal expression.

Article Snippet: Protein detection was performed using primary rabbit anti-human hepsin (Sigma Aldrich), phospho-Protein Kinase B (pAKT) (Invitrogen), phospho-extracellular signal-regulated kinases 1 and 2 (pERK1/2) (9101S, Cell Signaling Technologies, Werfen) and phospho-signal transducer and activator of transcription 3 (pSTAT3; Tyr705) (9145, Cell Signaling Technologies, Werfen) antibodies.

Techniques: Expressing, Electrophoresis, Western Blot, Over Expression

Journal:

Article Title: JAK2, But Not Src Family Kinases, Is Required for STAT, ERK, and Akt Signaling in Response to Growth Hormone in Preadipocytes and Hepatoma Cells

doi: 10.1210/me.2008-0015

Figure Lengend Snippet: Effect of Src Kinase Inhibitors on GH Activation of ERKS 1 and 2 and Akt

Article Snippet: Antibodies recognizing phosphor-STAT3 (αpY705-STAT3, catalog no. 9131), total STAT3 (αSTAT3, catalog no. 4904), phosphor-ERKs 1 and 2 (αpT202/pY204-ERK1/2, catalog no. 9106), total ERKs 1 and 2 (αERK1/2, catalog no. 4695), phosphor-Akt (αpS473-Akt, catalog no. 4058), total Akt (αAkt, catalog no. 9272), and phosphor-Src (αpY416-Src, catalog no. 2113) were from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay

Journal:

Article Title: JAK2, But Not Src Family Kinases, Is Required for STAT, ERK, and Akt Signaling in Response to Growth Hormone in Preadipocytes and Hepatoma Cells

doi: 10.1210/me.2008-0015

Figure Lengend Snippet: GH Activates JAK2, STATs 1, 3, and 5, ERKs 1 and 2, and Akt in SYF MEF Cells

Article Snippet: Antibodies recognizing phosphor-STAT3 (αpY705-STAT3, catalog no. 9131), total STAT3 (αSTAT3, catalog no. 4904), phosphor-ERKs 1 and 2 (αpT202/pY204-ERK1/2, catalog no. 9106), total ERKs 1 and 2 (αERK1/2, catalog no. 4695), phosphor-Akt (αpS473-Akt, catalog no. 4058), total Akt (αAkt, catalog no. 9272), and phosphor-Src (αpY416-Src, catalog no. 2113) were from Cell Signaling Technology (Danvers, MA).

Techniques:

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: Detection of LRRC26 protein in Lrrc26 message-rich mouse tissues. (A) Across the Top, membrane protein samples were prepared from each of the indicated tissues and immunoprecipitated with the LRRC26 polyclonal Ab. Samples were split to allow treatment with (+) or without (−) PNGase F, and also to allow detection of protein levels in the sample (across the Bottom). A Novus Anti-Na+/K+ATPaseA1 (ATP1A1) Ab was used to determine that similar amounts of protein from wt and LRRC26 KO samples were loaded. Protein amounts (in milligrams) used for LRRC26-IP product preparation were 0.1, 0.2, and 0.8 for lacrimal, parotid, and submandibular glands. For ATP1A1 detection, 75, 17, and 27 μg membrane proteins were loaded in each case. (B) For trachea, lactating mammary gland, and lung, the membrane protein amounts used to generate IP product were 0.5, 3.7, and 1.5 mg, with 55, 86, and 83 μg of membrane protein used to monitor ATP1A1. (C) For colon, glandular stomach, and cerebellum, 1.5, 1.1, and 1.6 mg of membrane protein samples were used for the preparation of IP product in each lane, whereas 44, 71, and 1 μg of membrane proteins were loaded for detection of ATP1A1 for the same tissues.

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: Immunoprecipitation

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: Bluo-Gal staining is observed in glandular acinar cells and goblet and Paneth cells of gastrointestinal tract. In A–D, tissues were developed for Bluo-Gal staining and counterstained with eosin. (A) LRRC26 KO tissues show abundant Bluo-Gal reaction product throughout a lacrimal gland section that is absent in wt sections. (B) In parotid, Bluo-Gal reaction product (Right) is observed sparsely, but throughout acinar cells, whereas dense staining likely corresponds to intralobular and interlobular parotid ducts. (C) Bluo-Gal staining in submandibular gland is confined to cells likely to be seromucous acinar cells, whereas larger glandular duct cells display little if any staining. (D) Bluo-Gal staining in sublingual gland is distributed throughout acinar cells and ducts within the gland. In E–G, tissues were first processed with Bluo-Gal staining followed by a periodic acid-Schiff (PAS) reaction. (E) Goblet cells in villi of the small intestine are positive for PAS staining at the apical end corresponding to mucus granules and positive for Bluo-Gal staining at the basal end. (F) Crypts in the colon exhibit abundant PAS-positive cells, with the most superficial cells also enriched with Bluo-Gal staining. (G) Crypts of the small intestine reveal positive PAS staining and Bluo-Gal staining in Paneth cells and goblet cells.

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: Staining

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: 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: KO of LRRC26 shifts gating of lacrimal gland and parotid cell BK currents rightward. (A1) Currents (shown to +140 mV) were activated with the indicated voltage protocol in a wt lacrimal gland cell. Pipette/intracellular Ca2+ was 250 nM for all whole-cell recordings. (A2) Currents are shown from a LRRC26 KO lacrimal gland cell with steps to +200 mV. (A3) Currents are shown up to +140 mV from a β4-KO lacrimal gland cell. For all panels in A, red traces are at +100 mV. (B) Panels on the Left (B1), Middle (B2), and Right (B3) correspond to currents from wt, LRRC26-KO, and β4-KO parotid gland cells, activated by voltage protocols identical to those in A. (C) GV curves were generated from tail currents in lacrimal gland cells from wt (n = 4), LRRC26 KO (n = 4), and β4 KO (n = 3). For wt and β4 KO, tail currents were measured at −20 mV, and for LRRC26-KO, +20 mV. Vh and z values from means of fits to individual cells are: for wt, Vh = 36.7 ± 1.4 mV with z = 1.2 + 0.1e; for LRRC26 KO, Vh = 182.2 ± 9.5 mV, z = 1.1 ± 0.1e; for β4 KO, Vh = 30.0 ± 8.8 mV, z = 1.2 ± 0.02e. (D) GV curves were generated from tail currents in parotid cells as in C. From fits to individual cells, for 12 wt cells, mean Vh = 33.5 ± 1.5 mV and z = 1.6 ± 0.1e; for 12 LRRC26 KO cells, Vh = 163.2 ± 6.7 mV with z = 1.3 ± 0.1e. Fit of averaged GVs yielded similar values: for wt, Vh = 33 with z = 1.3e, and for LRRC26 KO, Vh = 169.5, with z = 1.0e. (E) Traces show inhibition at +120 mV (maximal BK activation) by 5 mM TEA and 100 nM paxilline (Pax) in a wt parotid cell. (F) Traces shown inhibition at +200 mV (maximal activation) by 5 mM TEA and 100 nM paxilline in a LRRC26 KO parotid cell. (G) The mean % of current remaining compared with control in 5 mM TEA or 100 nM paxilline is plotted for wt and LRRC26 KO parotid cells, with open circles corresponding to individual cells.

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: Transferring, Generated, Inhibition, Activation Assay

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