mst1 d8b9q  (Cell Signaling Technology Inc)

Journal: Journal of biomechanics

Article Title: Mesenchymal stem cells differentiate dynamic from static load through variable regulation of the Hippo pathway

doi: 10.1016/j.jbiomech.2026.113228

Figure Lengend Snippet: ( A ) Cell lysates from SS treated cells were immunoprecipitated for MST1/2 and immunoblotted for PP2Ac. Representative densitometry shows increased association between MST1/2 and PP2Ac, (n = 3, p < 0.05). ( B ) Pulling down lysates with AB to PP2Ac before MST1/2 IB confirmed this result. ( C ) DS reduced the association between PP2AC and MST1/2. ( D ) Cells were stained with anti-PP2Ac (red) showing increase in nuclear PP2Ac signal after SS, scale bar = 25 μm. Fluorescence intensity of nucleus/cytoplasm of PP2Ac confirmed a significant effect of SS, **** = p < 0.0001. ( E ) MST1/2 (green) showed unchanged cellular localization after strains.

Article Snippet: AMOT 130 (cat#: 50–173-1230, Fisher), YAP (D8H1X) (cat#: 14074S, Cell Signaling), Phospho-YAP-ser127 (cat# 13008S, Cell Signaling), PP2A-Cα/β (cat# sc-80665, Santa Cruz), LATS1 (cat# 3477S, Cell Signaling), Phospho-LATS1 (Ser909) (cat# 9157S, Cell Signaling), MST1 (D8B9Q) (cat#14946S, Cell Signaling), Phospho-MST1/2 (Thr183) (cat# PA540255 , Invitrogen), Phosphoserine antibody (cat# AB9332, Abcam), Tubulin (cat# sc-23949, Santa Cruz).

Techniques: Immunoprecipitation, Staining, Fluorescence

Journal: Cell reports

Article Title: Epinephrine inhibits PI3Kα via the Hippo kinases

doi: 10.1016/j.celrep.2023.113535

Figure Lengend Snippet: (A) Experimental schema. (B) Dendrogram of the human protein kinome that highlights the kinases selected for our screen. (C) Thin-layer chromatography (TLC)-autoradiography of [32P]PIP3 produced by recombinant PI3Kα complexes (p110α/p85α, 500 nM) after incubation with the indicated recombinant protein kinases at 10 (L [low]) or 100 nM (H [high]) concentrations or with enzyme storage buffer (–). (D) PIP2 kinase assays with PI3Kα after incubation with subset of the GCK family, performed as described in (C). (E) PIP2 kinase assays with the class I PI3K isoforms (PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ, 500 nM) after incubation with MST1. (D) and (E) are representative of two independent replicates.

Article Snippet: MST1 (D8B9Q) Rabbit mAb , Cell Signaling Technology , Cat#14946; RRID:AB_2798654.

Techniques: Thin Layer Chromatography, Autoradiography, Produced, Recombinant, Incubation

Journal: Cell reports

Article Title: Epinephrine inhibits PI3Kα via the Hippo kinases

doi: 10.1016/j.celrep.2023.113535

Figure Lengend Snippet: (A) (Top) Peptide phosphorylation by MST1 to characterize its substrate consensus motif. Positional scanning peptide arrays were utilized, where 22 residues (20 amino acids +2 PTM residues) were scanned across nine neighboring positions of the phospho-acceptor. The zero controls (right inset), consisting of serine only, threonine only, or a 1:1 mixture of both, were examined as phospho-acceptors. Phosphorylation was measured by autoradiography. (Bottom) Sequence logo of the substrate consensus motif of MST1 as determined on top. Letter height is proportional to favorability of corresponding amino acid. (B) p110α’s threonine residues scored by MST1’s position-specific scoring matrix obtained from (A). (C) Incubation of p110α(WT)/p85α and p110α(T1061A)/p85α with MST1. Top: autoradiography of [32P]PIP3 production by p110α. Bottom: immunoblots of total p110α and pT1061 p110α. (D) Incubation of MST1 or MST2 with increasing concentrations of neratinib, followed by incubation with PI3Kα. Top: autoradiography of [32P]PIP3 production by p110α. Second from top: autoradiography of T1061-modeled peptide substrate peptide phosphorylation by MST1 or MST2. Bottom: immunoblots of total p110α and pT1061. (E) Repeat of (D) using HGK and its specific inhibitor, DMX-5804. (F) Immunoblot of p110α (WT and T1061A) on Phos-tag gel after treatment with MST1 or HGK. (A)–(F) are representative of two independent replicates.

Article Snippet: MST1 (D8B9Q) Rabbit mAb , Cell Signaling Technology , Cat#14946; RRID:AB_2798654.

Techniques: Phospho-proteomics, Autoradiography, Sequencing, Incubation, Western Blot

Journal: Cell reports

Article Title: Epinephrine inhibits PI3Kα via the Hippo kinases

doi: 10.1016/j.celrep.2023.113535

Figure Lengend Snippet: (A) Immunoblot for the indicated proteins using lysates from serum starved primary mouse hepatocytes isolated from WT mice that were stimulated with vehicle (DMSO), FSK (20 μM), or FSK and the MST1/2 inhibitor XMU (10 μM). (B) Immunoblot for the indicated proteins using lysates from HEK293A parental cells or a line with CRISPR deletion of MST1 and MST2 (MST1/2 DKO) that were serum starved for 2 h and pre-treated with vehicle (DMSO) or the PKA inhibitor H-89 (10 μM) for 1 h before stimulating with FSK (20 μM) for 10 or 20 min. (C) The ECAR was monitored in HEK293A parental, MST1/2 DKO, and Last1DKO cells for 1 h. Arrows indicate injection of glucose (10 mM), FSK (20 μM), Oligo (1 μM), and 2DG (50 mM). N = 15 wells. Results are representative of 3 independent experiments. (D) The suppression (%) of ECAR following the addition of FSK (20 μM) using data from (C). N = 15. (E) Immunoblot for the indicated proteins using lysates from livers taken from animals ( Stk3 f/f ; Stk4 f/f , Alb-Cre −/− and Stk3 f/f ; Stk4 f/f , Alb-Cre +/− ) that were injected with vehicle (normal saline) or Epi (0.3 μg/g). (F) Quantification of the ratios of pT1061 to total p110α and pAKT to total AKT using band intensity from (E). In (C), (D), and (F) data are represented as mean ± SEMs. Comparisons made via two-way ANOVA with Sidak’s multiple comparisons post-test (D) and ANOVA with Turkey’s multiple comparisons post-test (F).

Article Snippet: MST1 (D8B9Q) Rabbit mAb , Cell Signaling Technology , Cat#14946; RRID:AB_2798654.

Techniques: Western Blot, Isolation, CRISPR, Injection, Saline

Journal: Cell reports

Article Title: Epinephrine inhibits PI3Kα via the Hippo kinases

doi: 10.1016/j.celrep.2023.113535

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MST1 (D8B9Q) Rabbit mAb , Cell Signaling Technology , Cat#14946; RRID:AB_2798654.

Techniques: Recombinant, Fluorescence, Isolation, Double Knockout, Mutagenesis, Plasmid Preparation, Software