Journal: The Journal of biological chemistry

Article Title: Characterization of an alternative splice variant of LKB1.

doi: 10.1074/jbc.M806153200

Figure Lengend Snippet: FIGURE 5. The effect of forskolin treatment on LKB1 and AMPK activity. CCL13 cells were transiently transfected with FLAG-tagged human LKB1L, LKB1S, or catalytically inactive LKB1 harboring a D194A mutation (LKB1D194A). Prior to lysis, the cells were treated in the presence or absence of 20 M forskolin for 30 min, or 0.5 mM H2O2 for 5 min. A, a nuclear enriched fraction (20–50g)wasblottedwithanti-phospho-CREB(Ser-133),orcelllysates(50– 100 g) were blotted with anti-phospho-LKB1 (Ser-428) or anti-FLAG. The blots shown are representative of blots obtained from three independent experiments. B, LKB1 activity in anti-FLAG immune complexes isolated from 50 g of total lysate protein was determined by activation of recombinant AMPK. The results are the means S.E. of three independent experiments and are plotted as units/mg total lysate protein where 1 unit is the activity of LKB1 required to activate recombinant AMPK by 1 nmol/min/mg. C, endoge- nous AMPK was immunoprecipitated from 50 g of total protein using an anti-pan antibody bound to protein A-Sepharose. AMPK activity in the immune complexes was determined using the SAMS peptide assay. The results are plotted as pmol/min/mg and are the means S.E. of three inde- pendent experiments.

Article Snippet: Mouse monoclonal antibody recognizing both LKB1L and LKB1S (Ley37D/G6) and anti-STRAD (STRAD N13) were from Santa Cruz Biotechnology.Mousemonoclonal antibody recognizing Ser(P)-428 in human LKB1L, monoclonal anti-MO25, and phospho-CREB (Ser-133, 87G3) were from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Mutagenesis, Lysis, Isolation, Activation Assay, Recombinant, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Characterization of an alternative splice variant of LKB1.

doi: 10.1074/jbc.M806153200

Figure Lengend Snippet: FIGURE 7. Subcellular Localization of LKB1L and LKB1S. A, CCL13 cells were transiently transfected with FLAG-tagged human LKB1L or LKB1S alone (left panels) or co-transfected with STRAD and MO25 constructs (right panels). The cells were fixed in 4% paraformaldehyde and stained with a mouse monoclonal anti-LKB1 antibody (Ley37D/G6) that recognizes both LKB1L and LKB1S. Primary antibodies were detected with fluorescently linked secondary antibodies and visualized using a Leica TCS SP1 confocal microscope. B, HEK293 cells were treated with or without 20 M forskolin for 30 min and fractionated into cytosolic (C), membrane (M), and nuclear (N) enriched frac- tions. An equal volume of each fraction was blotted with an anti-LKB1 anti- body(Ley37D/G6).Thesamefractionswereusedtodeterminetheexpression of marker proteins for each of the fractions (Cytosolic, glyceraldehydes-3- phosphate dehydrogenase (GAPDH); Membrane, Na/K ATPase; Nuclear, CREB). C, cytosolic, membrane and nuclear fractions obtained from mouse testis were blotted with an anti-LKB1 antibody (Ley37D/G6). In each case, the blots shown are representative of blots obtained from three independent experiments. The migration of molecular mass markers is indicated.

Article Snippet: Mouse monoclonal antibody recognizing both LKB1L and LKB1S (Ley37D/G6) and anti-STRAD (STRAD N13) were from Santa Cruz Biotechnology.Mousemonoclonal antibody recognizing Ser(P)-428 in human LKB1L, monoclonal anti-MO25, and phospho-CREB (Ser-133, 87G3) were from Cell Signaling Technology.

Techniques: Transfection, Construct, Staining, Microscopy, Membrane, Marker, Migration

Journal: Cell reports

Article Title: SPTLC3 regulates plasma membrane sphingolipid composition to facilitate hepatic gluconeogenesis

doi: 10.1016/j.celrep.2024.115054

Figure Lengend Snippet:

Article Snippet: Phospho-CREB (Ser133) (87G3) Rabbit mAb , Cell Signaling Technology , RRID:AB_2561044.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: The Journal of Neuroscience

Article Title: β-Amyloid Peptide at Sublethal Concentrations Downregulates Brain-Derived Neurotrophic Factor Functions in Cultured Cortical Neurons

doi: 10.1523/JNEUROSCI.5463-03.2004

Figure Lengend Snippet: Pretreatment with sublethal concentrations of Aβ1-42 (5 or 10 μm) for 2 hr decreased the elevation of phosphorylated CREB levels induced by BDNF (50 ng/ml, 10 min). A, Western blot analysis of CREB phosphorylated at Ser133 (P-CREB) and total CREB (T-CREB). Aβ1-42 treatment resulted in a concentration-dependent decrease in the level of P-CREB but, considering all experiments (n = 3), had no significant effect on T-CREB levels. B, Quantification of the effect of pretreatment with 1, 5, or 10 μm Aβ1-42 (A1, A5, A10; A, here and in all other figures, stands for Aβ1-42). Estimates are the mean ± SEM (n = 3) expressed in terms of P-CREB levels obtained in the BDNF-exposed cultures (B, here and in all other figures, stands for BDNF). The effect of Aβ1-42 was significant (*p < 0.05, unpaired Student's t test). C, Pretreatment with 10 μm Aβ1-42 with random amino acid sequence [Aβ(R)] had no significant influence on the BDNF-induced increase of P-CREB levels. D, Immunohistochemical analysis of P-CREB. P-CREB immunoreactivity increased in BDNF-stimulated cultures (b) compared with unstimulated control (a). BDNF-induced increase in P-CREB immunoreactivity was suppressed by Aβ1-42 (5 μm) treatment (c). E, Exposure of the cultures to Aβ1-42 for 2 hr had no effect on P-CREB levels. In three experiments P-CREB levels in the presence of 5 and 10 μm Aβ1-42, respectively, were 104 ± 2 and 105 ± 6% of basal. F, Analysis of CRE-mediated transcriptional activity. Cortical neurons at 3 DIV were transfected with plasmid pCRE-Luc containing CRE sequences and a luciferase reporter gene (see Materials and Methods). Cells transfected with a CMV-luciferase control plasmid served to normalize CRE activity. After 40 hr the cultures were switched to fresh medium and incubated for 1 hr in the presence or the absence of 5 μm Aβ1-42. Transfected cortical cultures were incubated for an additional 9 hr either with or without the addition of 50 ng/ml BDNF, and transcriptional activity was measured by the luciferase assay. Aβ1-42 treatment decreased the BDNF-induced transcriptional activity of CRE. Estimates are the mean ± SEM (n = 3) expressed in terms of BDNF-induced transcriptional activity. The effect of Aβ1-42 was significant (*p < 0.05, unpaired Student's t test).

Article Snippet: Membranes were incubated additionally for 2 hr at room temperature in the presence of the following antibodies as indicated: from Upstate Cell Signaling (Charlottesville, VA), phosphorylated CREB (P-CREB; detects CREB phosphorylated at Ser 133 ; 1:2000), total CREB (T-CREB; 1:2000), Shc (the antibody recognizes all isoforms; 1:1000), PLCγ (1:1000), total TrkB (T-TrkB; 1:1000); from Cell Signaling Technology (Beverly, MA), phosphorylated MAPK/ERK (P-MAPK; detects p44/42 MAPK phosphorylated at Thr 202 and Tyr 204 ; 1:2000), total MAPK (T-MAPK; 1:2000), phosphorylated Raf-1 (P-Raf-1; detects Raf-1 phosphorylated at Ser 388 ; 1:1000), phosphorylated MEK1/2 (P-MEK1/2; detects MEK1/2 phosphorylated at Ser 217/221 ; 1:1000), total MEK1/2 (T-MEK1/2; 1:1000), phosphorylated Elk-1 (P-Elk-1; detects Elk-1 phosphorylated at Ser 383 ; 1:1000), total Elk-1 (T-Elk-1; 1:1000), phosphorylated Akt (P-Akt; detects Akt phosphorylated at Ser 473 ; 1:1000), total Akt (T-Akt; 1:1000), phosphorylated TrkB (P-TrkB; detects TrkB phosphorylated at Tyr 490 ; 1:500).

Techniques: Western Blot, Concentration Assay, Sequencing, Immunohistochemical staining, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Incubation