plcγ1 protein  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Disruption of Phosphoinositide-Specific Phospholipases Cγ1 Contributes to Extracellular Matrix Synthesis of Human Osteoarthritis Chondrocytes

doi: 10.3390/ijms150813236

Figure Lengend Snippet: The expression level of phosphoinositide-specific phospholipase γ1 (PLCγ1) in normal articular and OA chondrocytes. Normal samples were obtained from 3 patients with amputation from accident, and OA samples were obtained from 20 patients with advanced OA. ( A ) The protein expression levels of PLCγ1 and p-PLCγ1 in cultured normal and OA chondrocytes were detected with western blotting analysis using rat anti-PLC-γ1, p-PLC-γ1, and GAPDH antibodies according to Materials and Methods. The values represent the mean ±S.E.M. of five independent experiments, each yielding similar results ( * p < 0.05); ( B ) The protein expression level of PLCγ1 in normal articular and OA cartilage was detected with immunohistochemistry analysis according to Materials and Method (original magnification ×100 or ×400, * p < 0.05).

Article Snippet: Antibodies against PLCγ1 (CST#2822S,), p-PLCγ1-Tyr783 (CST#2821S), mTOR (CST#2983), p-mTOR-Ser2448 (CST#2983), p-p70S6K-Thr389 (CST#9234S), p-S6-Ser235/236 (CST#2211S), GAPDH (CST#2251-1), NF-κBp65 (CST#8242), and PLCγ1 siRNA (CST#6293) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Techniques: Expressing, Cell Culture, Western Blot, Immunohistochemistry

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Expression of PLCγ1 in MII-arrested mouse eggs, and schematic representation of the bacterial fusion proteins containing different PLCγ1 domains. In the schematic representation of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two antibodies used for microinjection experiments shown in Table . PH , pleckstrin homology domain; Ca 2 + , EF-hand domain (calcium binding motif); X and Y , split catalytic domain; P and H , split pleckstrin homology domain; SH2 and SH3 , src-homology domains; C2 , Ca 2+ -dependent lipid binding domain. ( A ) Western blot from extracts of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50 mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods section, and used for the microinjection experiments shown in Table . ( B ) Bacterial fusion proteins containing different PLCγ1 domains used for the experiments shown in Table . The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1 , GST; lane 2 , GST-PLCγ1-SH2SH2SH3; lane 3 , GST-PLCγ1-SH2SH2; lane 4 , GST-PLCγ1-SH3.

Article Snippet: Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. ) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

Techniques: Expressing, Microinjection, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Staining, SDS Page, Affinity Purification

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: A GST-PLCγl-SH3 Domain Fusion Protein Specifically Inhibits Tr-kit–induced Parthenogenetic Activation of Mouse Eggs

Article Snippet: Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. ) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

Techniques: Activation Assay

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical granules exocytosis and pronuclear formation in mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit and 500 μg/ml (final concentration in the egg: ∼10 μg/ml) of either GST-PLCγ1-SH3 ( A ) or GST-PLCγ1-SH2SH2 ( B ) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a similar rate as for pronuclear formation (see Table ) in three separate experiments. Double staining of chromatin (Hoechst dye) and cortical granules (TRITC-labeled lectin) was performed as described under Materials and Methods. Bar, 30 μm.

Article Snippet: Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. ) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

Techniques: Injection, Recombinant, Concentration Assay, Double Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Antibodies Against the SH Region of PLCγ1 Inhibit Tr-kit–induced Parthenogenetic Activation of Mouse Eggs

Article Snippet: Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. ) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

Techniques: Activation Assay

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Tr-kit stimulates DAG and InsP production in COS cells coexpressing PLCγ1. Cells were transfected with the indicated expression vectors and labeled with either [ 3 H]arachidonic acid ( A ) or [ 3 H]inositol ( B ) and processed as described under Materials and Methods. ( A ) DAG content was measured as cpm incorporated in DAG versus 10 3 cpm incorporated in total lipids. ( B ) InsPs content was measured as cpm incorporated into InsPs per mg protein 24 h after transfection. The data represent the mean ± SD of three separate experiments, each performed in triplicate. ( C ) Pellets obtained after TCA precipitation of representative samples analyzed for InsP production were resuspended in SDS-PAGE sample buffer and analyzed in Western blot (50 μg in each lane) by using either anti-PLCγ1bd or anti-kit antibodies.

Article Snippet: Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. ) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

Techniques: Transfection, Expressing, Labeling, TCA Precipitation, SDS Page, Western Blot

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Tr-kit does not stably associate with PLCγ1. ( A ) COS cells were transfected with no DNA ( mock ), or 20 μg/dish pCMV5-c-kit ( c-kit ), or 20 μg/dish pCMV5-tr-kit ( tr-kit ). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody ( right side of the panel ), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody ( left side of the panel ). ( B ) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. ( C ) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.

Article Snippet: Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. ) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

Techniques: Stable Transfection, Transfection, Incubation, Western Blot, Expressing, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Tr-kit stimulates tyrosine phosphorylation of PLCγ1 in transfected COS cells. Cells were transfected with a PLCγ1 expression vector, either alone or together with the tr-kit expression vector. ( A ) Cell extracts were immunoprecipitated using a mixture of anti-PLCγ1 antibodies (see Materials and Methods). Immunoprecipitated proteins were analyzed in Western blot using either the anti-PLCγ1bd antibody ( αPLCγ1 , left panel ) or an anti-phosphotyrosine antibody ( αPY , right panel ). These images are representative of six separate experiments, which gave similar results. ( B ) Western blot analysis with anti-PLCγ1 antibody ( αPLCγ1 ) and anti-phosphotyrosine antibody ( αPY ) of total cell extracts (50 μg in each lane) from PLCγ1- and PLCγ1/tr-kit– transfected COS cells.

Article Snippet: Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. ) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

Techniques: Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Expression of PLCγ1 in MII-arrested mouse eggs, and schematic representation of the bacterial fusion proteins containing different PLCγ1 domains. In the schematic representation of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two antibodies used for microinjection experiments shown in Table . PH , pleckstrin homology domain; Ca 2 + , EF-hand domain (calcium binding motif); X and Y , split catalytic domain; P and H , split pleckstrin homology domain; SH2 and SH3 , src-homology domains; C2 , Ca 2+ -dependent lipid binding domain. ( A ) Western blot from extracts of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50 mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods section, and used for the microinjection experiments shown in Table . ( B ) Bacterial fusion proteins containing different PLCγ1 domains used for the experiments shown in Table . The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1 , GST; lane 2 , GST-PLCγ1-SH2SH2SH3; lane 3 , GST-PLCγ1-SH2SH2; lane 4 , GST-PLCγ1-SH3.

Article Snippet: Affinity-purified GST-PLCγ1 SH region fusion protein (GST-PLCγ1-SH2SH2SH3) (No. sc4019), and GST-Grb2-SH3 (residues 156–199; No. sc4036) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Staining, SDS Page, Affinity Purification

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical granules exocytosis and pronuclear formation in mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit and 500 μg/ml (final concentration in the egg: ∼10 μg/ml) of either GST-PLCγ1-SH3 ( A ) or GST-PLCγ1-SH2SH2 ( B ) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a similar rate as for pronuclear formation (see Table ) in three separate experiments. Double staining of chromatin (Hoechst dye) and cortical granules (TRITC-labeled lectin) was performed as described under Materials and Methods. Bar, 30 μm.

Article Snippet: Affinity-purified GST-PLCγ1 SH region fusion protein (GST-PLCγ1-SH2SH2SH3) (No. sc4019), and GST-Grb2-SH3 (residues 156–199; No. sc4036) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Injection, Recombinant, Concentration Assay, Double Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Antibodies Against the SH Region of PLCγ1 Inhibit Tr-kit–induced Parthenogenetic Activation of Mouse Eggs

Article Snippet: Affinity-purified GST-PLCγ1 SH region fusion protein (GST-PLCγ1-SH2SH2SH3) (No. sc4019), and GST-Grb2-SH3 (residues 156–199; No. sc4036) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Tr-kit stimulates DAG and InsP production in COS cells coexpressing PLCγ1. Cells were transfected with the indicated expression vectors and labeled with either [ 3 H]arachidonic acid ( A ) or [ 3 H]inositol ( B ) and processed as described under Materials and Methods. ( A ) DAG content was measured as cpm incorporated in DAG versus 10 3 cpm incorporated in total lipids. ( B ) InsPs content was measured as cpm incorporated into InsPs per mg protein 24 h after transfection. The data represent the mean ± SD of three separate experiments, each performed in triplicate. ( C ) Pellets obtained after TCA precipitation of representative samples analyzed for InsP production were resuspended in SDS-PAGE sample buffer and analyzed in Western blot (50 μg in each lane) by using either anti-PLCγ1bd or anti-kit antibodies.

Article Snippet: Affinity-purified GST-PLCγ1 SH region fusion protein (GST-PLCγ1-SH2SH2SH3) (No. sc4019), and GST-Grb2-SH3 (residues 156–199; No. sc4036) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Expressing, Labeling, TCA Precipitation, SDS Page, Western Blot

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Tr-kit does not stably associate with PLCγ1. ( A ) COS cells were transfected with no DNA ( mock ), or 20 μg/dish pCMV5-c-kit ( c-kit ), or 20 μg/dish pCMV5-tr-kit ( tr-kit ). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody ( right side of the panel ), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody ( left side of the panel ). ( B ) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. ( C ) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.

Article Snippet: Affinity-purified GST-PLCγ1 SH region fusion protein (GST-PLCγ1-SH2SH2SH3) (No. sc4019), and GST-Grb2-SH3 (residues 156–199; No. sc4036) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Stable Transfection, Transfection, Incubation, Western Blot, Expressing, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:

Figure Lengend Snippet: Tr-kit stimulates tyrosine phosphorylation of PLCγ1 in transfected COS cells. Cells were transfected with a PLCγ1 expression vector, either alone or together with the tr-kit expression vector. ( A ) Cell extracts were immunoprecipitated using a mixture of anti-PLCγ1 antibodies (see Materials and Methods). Immunoprecipitated proteins were analyzed in Western blot using either the anti-PLCγ1bd antibody ( αPLCγ1 , left panel ) or an anti-phosphotyrosine antibody ( αPY , right panel ). These images are representative of six separate experiments, which gave similar results. ( B ) Western blot analysis with anti-PLCγ1 antibody ( αPLCγ1 ) and anti-phosphotyrosine antibody ( αPY ) of total cell extracts (50 μg in each lane) from PLCγ1- and PLCγ1/tr-kit– transfected COS cells.

Article Snippet: Affinity-purified GST-PLCγ1 SH region fusion protein (GST-PLCγ1-SH2SH2SH3) (No. sc4019), and GST-Grb2-SH3 (residues 156–199; No. sc4036) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot