anti-phospho-mertk antibody  (PhosphoSolutions)

Journal: Cancers

Article Title: Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition

doi: 10.3390/cancers16122253

Figure Lengend Snippet: TAM/Met receptor tyrosine kinases are upregulated in TNBC. ( a ) Schematic representation of receptor tyrosine kinase-mediated regulation of CDK4/6. ( b , c ) Immunoblot was performed on cell lines treated for 24 h with Abe (2 μM) ( b ) and for 25 min with either HGF (40 ng/mL) or Gas6 (400 ng/mL) ( c ). Protein levels were determined for phospho-AXL and phospho−MET. ( d ) Comparison of gene expression levels in TNBC vs. non-TNBC, based on RNAseq data from breast cancer patients. ( e ) TMA IHC staining for total Axl, Met, and MerTK in TNBC and HER2+ breast cancer (lower panel). Scale bars are 0.5 mm for 2.5× and 50 μm for 20×. Violin plots show the quantification of each protein expression based on the H-scoring in TNBC vs. HER2+ (two-tailed t -test). ( f ) The Kaplan–Meier survival estimate for MerTK, Met, and Axl based on the RNAseq data from all breast cancer patients. Abe: abemaciclib. The original western blot figures can be found in File S1.

Article Snippet: The following antibodies were used for immunoblotting: phospho-Met (Tyr1234/1235) (CST, 3077), Met (D1C2) (CST, 8198), Axl (C89E7) (CST, 8661), phospho-Axl (Y779) (R&D Systems, MAB6965), phospho-MerTK (Phosphosolutions, Denver, CO, USA, p186-749), MerTK (Abcam, Cambridge, UK, ab52968), phospho-Akt (CST, 9271), phospho-mTOR (abclonal, AP0094), and ERBB2 (CST, 2165).

Techniques: Western Blot, Comparison, Gene Expression, Immunohistochemistry, Expressing, Two Tailed Test

Journal: Cancers

Article Title: Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition

doi: 10.3390/cancers16122253

Figure Lengend Snippet: The combination of sitravatinib with abemaciclib or palbociclib is highly toxic against TNBC cells. ( a ) Chemical structure of sitravatinib (Sitra). ( b ) Immunoblot was performed on cell lines treated for 24 h with Abe (2 μm), Palbo (5 μm), and/or Sitra (2 μm). Protein levels were determined for phospho-AXL, phosho-MET, and phosho-MERTK. ( c ) The clonogenic assay showing that the combination of Abe or Palbo with Sitra significantly decreased the colony formation capacity of TNBC cells. Representative images of stained colonies. ( d ) Combination index (CI) values for the combinations of sitravatinib or merestinib with CDK4/6 inhibitor abemaciclib using different doses. Circles represent experimentally determined CI values using the Chou–Talalay method. The colors (orange and blue) represent the fixed ratio mixtures. ( e , f ) Overview of the toxicity and synergy scores of the drug combinations for TNBC lines. The heatmaps show the level of toxicity ( e ) and Bliss number ( f ) for the cell lines tested in this study. Average values of toxicity ( e ) or Bliss number ( f ) for cells treated with sitravatinib (S) at varying doses (S0 = No Drug, S1 = 1 μm, S2 = 2 μm, and S3 = 3 μm) in combination with either abemaciclib (A) at varying doses (A0 = No Drug, A1 = 1 μm, A2 =2 μm, A3 = 3 μm, and A4 = 4 μm) or palbociclib at varying doses (P0 = No Drug, P1 = 1 μm, P2 = 2 μm, P3 = 3 μm, and P4 = 4 μm). ( g ) Shown is the caspase-3/7 activity measured upon 24 h of drug treatments. The data are presented as mean ± SEM from three independent experiments, expressed as ratios to untreated control values, with associated p values as indicated (One-way ANOVA with Dunnett’s multiple comparisons test analysis). Abe: abemaciclib; Palbo: palbociclib. The original western blot figures can be found in File S1.

Article Snippet: The following antibodies were used for immunoblotting: phospho-Met (Tyr1234/1235) (CST, 3077), Met (D1C2) (CST, 8198), Axl (C89E7) (CST, 8661), phospho-Axl (Y779) (R&D Systems, MAB6965), phospho-MerTK (Phosphosolutions, Denver, CO, USA, p186-749), MerTK (Abcam, Cambridge, UK, ab52968), phospho-Akt (CST, 9271), phospho-mTOR (abclonal, AP0094), and ERBB2 (CST, 2165).

Techniques: Western Blot, Clonogenic Assay, Staining, Activity Assay, Control

Journal: Cancers

Article Title: Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition

doi: 10.3390/cancers16122253

Figure Lengend Snippet: Lapatinib-resistant HER2+ cell lines became more sensitive to the combination of sitravatinib with abemaciclib or palbociclib. ( a ) Overview of the toxicity of the drug combinations for HER2+ cell lines. The heatmaps show the level of toxicity for the cell lines tested. Average values of toxicity for cells treated with sitravatinib (S) at varying doses (S0 = No Drug, S1 = 1 μm) in combination with either abemaciclib (A) (A0 = No Drug, A1 = 1 μm, and A2 = 2 μm) or palbociclib (P0 = No Drug, P1 = 1 μm, and P2 = 2 μm). ( b ) The clonogenic assay showing that the combination of Abe or Palbo with Sitra had only modest effect on the HER2+ cell line SKBR3. Representative images of stained colonies. ( c ) Schematic representation of the generation of lapatinib-resistant (LapR) HER2 lines through continuous lapatinib treatment with gradual increase in treatment dose up to 30 μm. Cell viability confirming the resistance of the LapR cells to high doses of lapatinib (30 μm). ( d , e ) qRT-PCR and immunoblot showing increased expressions of Axl, Met, and MerTK with the suppression of Her2 levels in LapR vs. the parental cells. ( f ) Cell viability showing increased sensitivity of SKBR3 LapR cells to the combination of abemaciclib or palbociclib with sitravatinib compared with the parental SKBR3 cells. Overview of the toxicity of the drug combinations for HER2+ and LapR HER2 cell lines. The heatmaps show the level of toxicity for the cell lines tested. Average values of toxicity for cells treated with sitravatinib (S) at varying doses (S0 = No Drug, S1 = 1 μm, and S2 = 2 μm) in combination with either abemaciclib (A) (A0 = No Drug, A1 = 1 μm, A2 = 2 μm, and A3 = 3 μm) or palbociclib (P0 = No Drug, P1 = 1 μm, and P2 = 2 μm). ( g ) The clonogenic assay showing that SKBR3-LapR cells became highly sensitive to the combination of Abe or Palbo with Sitra. Representative images of stained colonies. Abe: abemaciclib; Palbo: palbociclib; Sitra: sitravatinib. Each bar represents mean ± SEM from three independent experiments, with associated p (* p < 0.05, *** p < 0.0001; one-way ANOVA with post hoc Tukey analysis). The original western blot figures can be found in File S1.

Article Snippet: The following antibodies were used for immunoblotting: phospho-Met (Tyr1234/1235) (CST, 3077), Met (D1C2) (CST, 8198), Axl (C89E7) (CST, 8661), phospho-Axl (Y779) (R&D Systems, MAB6965), phospho-MerTK (Phosphosolutions, Denver, CO, USA, p186-749), MerTK (Abcam, Cambridge, UK, ab52968), phospho-Akt (CST, 9271), phospho-mTOR (abclonal, AP0094), and ERBB2 (CST, 2165).

Techniques: Clonogenic Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: Aging (Albany NY)

Article Title: Myeloid-specific knockout of SHP2 regulates PI3K/PLCγ signaling pathway to protect against early myocardial infarction injury

doi: 10.18632/aging.205096

Figure Lengend Snippet: SHP2 MAC-KO can affect the expression of PI3K/PLCγ signaling pathway, the expression of inflammatory factors and proteins related to phagocytic activity. ( A ) Protein band diagram of p-SHP2, p-PI3K, p-MerTK, p-PLCγ2, p-MyD88, p-NF-κB, TNFα, IFNγ, IL-1β; ( B ) Relative protein expression of p-SHP2, p-PI3K, p-MerTK, p-PLCγ2, p-MyD88, p-NF-κB, TNFα, IFNγ, IL-1β. **P<0.01.

Article Snippet: Then the membrane was transferred, and put in the TBST buffer containing 5% skim milk powder, blocked for 2 h. Primary antibodies p-SHP2(Abcam, ab62322, 1:1000), p-PI3K (CellSignaling, 17366, 1:1000), p-MerTK (CellSignaling, 44463,1:1000), p-PLCγ2 (CellSignaling, 3871, 1:1000), p-Myd88 (CellSignaling, 17366, 1:1000), p-NF-κB (CellSignaling, 3303,1:1000), TNFα (CellSignaling, 11948, 1:1000), IFNγ (CellSignaling, 8455, 1:1000), IL-1β (CellSignaling, 12703, 1:1000), and GADPH (CellSignaling, 5174, 1:1000) were added, and were incubated at room temperature for 1 hour, then incubated at 4° C overnight.

Techniques: Expressing, Activity Assay

Journal: Aging (Albany NY)

Article Title: Myeloid-specific knockout of SHP2 regulates PI3K/PLCγ signaling pathway to protect against early myocardial infarction injury

doi: 10.18632/aging.205096

Figure Lengend Snippet: SHP2 MAC-KO can mediate the PI3K/PLCγ signaling pathway to influence the expression of inflammatory factors and proteins related to phagocytic activity. ( A ) Protein band diagram of p-PI3K, p-MerTK, p-PLC Γ2, p-MyD88, p-NF-κb, TNF α, IFN γ, il-1β; ( B ) Relative protein expression amounts of p-PI3K, p-MerTK, p-PLCγ2, p-MyD88, p-NF-κb, TNF α, IL-1β. **P<0.01, ns P>0.05.

Article Snippet: Then the membrane was transferred, and put in the TBST buffer containing 5% skim milk powder, blocked for 2 h. Primary antibodies p-SHP2(Abcam, ab62322, 1:1000), p-PI3K (CellSignaling, 17366, 1:1000), p-MerTK (CellSignaling, 44463,1:1000), p-PLCγ2 (CellSignaling, 3871, 1:1000), p-Myd88 (CellSignaling, 17366, 1:1000), p-NF-κB (CellSignaling, 3303,1:1000), TNFα (CellSignaling, 11948, 1:1000), IFNγ (CellSignaling, 8455, 1:1000), IL-1β (CellSignaling, 12703, 1:1000), and GADPH (CellSignaling, 5174, 1:1000) were added, and were incubated at room temperature for 1 hour, then incubated at 4° C overnight.

Techniques: Expressing, Activity Assay