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Santa Cruz Biotechnology grb7 (n-20) n-terminal grb7, rabbit polyclonal sc-607 antibody

<t>Grb7</t> expression in the mouse embryo visualised by immunohistochemistry on wild type e14.5 paraffin sections. Representative mid-sagittal sections were chosen to display a wide range of tissues (sections adjacent to those used to stain for Grb14 in Fig. ). A Whole wild type embryo with expression highlighted in dermis (d), gut (g), inner ear (i), liver (li), lung (lu), nasal epithelium (ne), pancreas (p), pituitary (pi), ribs (r, ossified cartilage), stomach (s), salivary gland (sg) and tooth primordia (tp); B Expression in submandibular gland and tooth primordia; C Expression in kidney (ki) and adrenal gland (ad); D Expression in lung and liver, but not heart (h); E Expression in epithelial lining of mid- and hind-gut; F Endocrine pancreas. Brown staining is indicative of Grb7 expression, magnifications as indicated

Grb7 (N 20) N Terminal Grb7, Rabbit Polyclonal Sc 607 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Grb7 , Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism"

Article Title: Grb7 , Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Grb7 expression in the mouse embryo visualised by immunohistochemistry on wild type e14.5 paraffin sections. Representative mid-sagittal sections were chosen to display a wide range of tissues (sections adjacent to those used to stain for Grb14 in Fig. ). A Whole wild type embryo with expression highlighted in dermis (d), gut (g), inner ear (i), liver (li), lung (lu), nasal epithelium (ne), pancreas (p), pituitary (pi), ribs (r, ossified cartilage), stomach (s), salivary gland (sg) and tooth primordia (tp); B Expression in submandibular gland and tooth primordia; C Expression in kidney (ki) and adrenal gland (ad); D Expression in lung and liver, but not heart (h); E Expression in epithelial lining of mid- and hind-gut; F Endocrine pancreas. Brown staining is indicative of Grb7 expression, magnifications as indicated

Figure Legend Snippet: Grb7 expression in the mouse embryo visualised by immunohistochemistry on wild type e14.5 paraffin sections. Representative mid-sagittal sections were chosen to display a wide range of tissues (sections adjacent to those used to stain for Grb14 in Fig. ). A Whole wild type embryo with expression highlighted in dermis (d), gut (g), inner ear (i), liver (li), lung (lu), nasal epithelium (ne), pancreas (p), pituitary (pi), ribs (r, ossified cartilage), stomach (s), salivary gland (sg) and tooth primordia (tp); B Expression in submandibular gland and tooth primordia; C Expression in kidney (ki) and adrenal gland (ad); D Expression in lung and liver, but not heart (h); E Expression in epithelial lining of mid- and hind-gut; F Endocrine pancreas. Brown staining is indicative of Grb7 expression, magnifications as indicated

Techniques Used: Expressing, Immunohistochemistry, Staining

Grb14 expression in the mouse embryo visualised by immunohistochemistry on wild type e14.5 paraffin sections. Representative mid-sagittal sections were chosen to display a wide range of tissues (sections adjacent to those used to stain for Grb7 in Fig. ). A Whole wild type embryo with expression highlighted in cardiac muscle (c), choroid plexus (cp), dermis (d), diaphragm (di), gut (g), lungs (lu), liver (li), ribs (r, ossifying cartilage), stomach (s), tooth primordia (tp) and tongue (t); B Expression in choroid plexus ; C Expression in ossifying cartilage (ca) of vertebrae; D Expression in lung, ossifying cartilage of ribs and intercostal muscle (ic); E Expression in pancreas (p). Brown staining is indicative of Grb14 expression, magnifications as indicated

Figure Legend Snippet: Grb14 expression in the mouse embryo visualised by immunohistochemistry on wild type e14.5 paraffin sections. Representative mid-sagittal sections were chosen to display a wide range of tissues (sections adjacent to those used to stain for Grb7 in Fig. ). A Whole wild type embryo with expression highlighted in cardiac muscle (c), choroid plexus (cp), dermis (d), diaphragm (di), gut (g), lungs (lu), liver (li), ribs (r, ossifying cartilage), stomach (s), tooth primordia (tp) and tongue (t); B Expression in choroid plexus ; C Expression in ossifying cartilage (ca) of vertebrae; D Expression in lung, ossifying cartilage of ribs and intercostal muscle (ic); E Expression in pancreas (p). Brown staining is indicative of Grb14 expression, magnifications as indicated

Techniques Used: Expressing, Immunohistochemistry, Staining

Expression of Grb7 family members in PN21 mouse pancreas visualised by immunohistochemistry on paraffin sections. Antibodies specific for ( A ) Grb7; B Grb10; and C Grb14; were used to detect distinct proteins of the Grb7 family. Brown staining is indicative of protein expression. Arrows indicate endocrine area (pancreatic islets); magnification 100x

Figure Legend Snippet: Expression of Grb7 family members in PN21 mouse pancreas visualised by immunohistochemistry on paraffin sections. Antibodies specific for ( A ) Grb7; B Grb10; and C Grb14; were used to detect distinct proteins of the Grb7 family. Brown staining is indicative of protein expression. Arrows indicate endocrine area (pancreatic islets); magnification 100x

Techniques Used: Expressing, Immunohistochemistry, Staining

Genetic crosses used in the study, showing parent and offspring genotypes with their expected ratios. Crosses between (A) Grb10 +/p : Grb14 +/- double heterozygous females and Grb10 +/p : Grb14 +/- double heterozygous males, producing offspring of twelve genotypes, and (B) Grb7 +/- : Grb10 +/p double heterozygous females and Grb7 +/- heterozygous males, producing offspring of six genotypes, each in the indicated, expected Mendelian ratios. For ANOVA statistical analysis these six or twelve genotypes were used to form four groups, as indicated, since no difference was anticipated between animals differing only in their Grb14 +/- and Grb14 +/+ or Grb7 +/- and Grb7 +/+ allelic status. Similarly, due to the imprinted expression of Grb10 , offspring inheriting a mutant copy of the paternal Grb10 allele ( Grb10 +/p ), having no growth phenotype, were not expected to differ from Grb10 wild type ( Grb10 +/+ ) offspring, while double knockout (DKO) offspring inheriting mutations of both parental alleles ( Grb10 m/p ) were expected to be indistinguishable from those inheriting a mutant copy of the normally active maternal Grb10 allele ( Grb10 m/+ )

Figure Legend Snippet: Genetic crosses used in the study, showing parent and offspring genotypes with their expected ratios. Crosses between (A) Grb10 +/p : Grb14 +/- double heterozygous females and Grb10 +/p : Grb14 +/- double heterozygous males, producing offspring of twelve genotypes, and (B) Grb7 +/- : Grb10 +/p double heterozygous females and Grb7 +/- heterozygous males, producing offspring of six genotypes, each in the indicated, expected Mendelian ratios. For ANOVA statistical analysis these six or twelve genotypes were used to form four groups, as indicated, since no difference was anticipated between animals differing only in their Grb14 +/- and Grb14 +/+ or Grb7 +/- and Grb7 +/+ allelic status. Similarly, due to the imprinted expression of Grb10 , offspring inheriting a mutant copy of the paternal Grb10 allele ( Grb10 +/p ), having no growth phenotype, were not expected to differ from Grb10 wild type ( Grb10 +/+ ) offspring, while double knockout (DKO) offspring inheriting mutations of both parental alleles ( Grb10 m/p ) were expected to be indistinguishable from those inheriting a mutant copy of the normally active maternal Grb10 allele ( Grb10 m/+ )

Techniques Used: Expressing, Mutagenesis, Double Knockout

Summary of PN1 body and organ weight data for progeny of crosses between (A) the Grb10 KO and Grb14 KO strains, and (B) the Grb7 KO and Grb10 KO strains. Mean weights are shown for each genotype together with changes relative to wild type (%WT) for each mutant genotype

Figure Legend Snippet: Summary of PN1 body and organ weight data for progeny of crosses between (A) the Grb10 KO and Grb14 KO strains, and (B) the Grb7 KO and Grb10 KO strains. Mean weights are shown for each genotype together with changes relative to wild type (%WT) for each mutant genotype

Techniques Used: Mutagenesis

Generation of a Grb7 KO mouse strain. A null mutation was designed, lacking all the protein coding sequence. A The wild type Grb7 locus (top), showing exons (filled boxes numbered 1–15), translational start (open triangle) and stop (filled triangle) codons, plus selected restriction enzyme sites. Note that the translational start (ATG) codon lies within a Nco I restriction site (CCATGG) used as part of the cloning strategy. Regions used to direct homologous recombination in ES cells (5’ arm and 3’ arm) and also probes used to confirm correct targeting are indicated to either side of the coding exons. The homologous arms were cloned into the pPNT vector to form the pPNT- Grb7 targeting construct (middle), in which the coding exons have been replaced with a neomycin ( neo ) resistance gene cassette for positive selection of ES cells stably incorporating the construct. The targeting construct also includes a herpes simplex virus thymidine kinase ( hsv-tk ) gene, located outside the homologous regions that is typically retained at sites of random integration, and was used to enrich for targeting events by negative selection. Consequently, the correctly targeted allele (bottom) retains the neo but not the hsv-tk gene. B Southern blot analysis of Hind III digested DNA from wild type ES cells (ES) or primary mouse embryonic fibroblasts (MEF) alongside three successfully targeted ES cell clones (2E5, 4B5 and 5D1). In the targeted (TG) allele, loss of the sequence between the homologous arms alters the distance between restriction enzyme sites, compared with the wild type (WT) allele. Consequently, in the wild type allele Probe A recognises a 5′ 8.7 kb fragment and Probe B a 3′ 10.8 kb fragment, whereas both probes detect a15.3 kb fragment for the targeted allele. C Western blot analysis of protein extracts from adult kidney and liver derived from Grb7 wild type (+ / +) and Grb7 KO (-/-) homozygous mice following establishment of a true breeding line. The blot was probed with an antibody specific for Grb7, that readily detects a species of approximately the correct size (65 kDa) in wild type but not Grb7 KO samples, whereas an antibody specific for α-tubulin (50 kDa) was detected equally in both

Figure Legend Snippet: Generation of a Grb7 KO mouse strain. A null mutation was designed, lacking all the protein coding sequence. A The wild type Grb7 locus (top), showing exons (filled boxes numbered 1–15), translational start (open triangle) and stop (filled triangle) codons, plus selected restriction enzyme sites. Note that the translational start (ATG) codon lies within a Nco I restriction site (CCATGG) used as part of the cloning strategy. Regions used to direct homologous recombination in ES cells (5’ arm and 3’ arm) and also probes used to confirm correct targeting are indicated to either side of the coding exons. The homologous arms were cloned into the pPNT vector to form the pPNT- Grb7 targeting construct (middle), in which the coding exons have been replaced with a neomycin ( neo ) resistance gene cassette for positive selection of ES cells stably incorporating the construct. The targeting construct also includes a herpes simplex virus thymidine kinase ( hsv-tk ) gene, located outside the homologous regions that is typically retained at sites of random integration, and was used to enrich for targeting events by negative selection. Consequently, the correctly targeted allele (bottom) retains the neo but not the hsv-tk gene. B Southern blot analysis of Hind III digested DNA from wild type ES cells (ES) or primary mouse embryonic fibroblasts (MEF) alongside three successfully targeted ES cell clones (2E5, 4B5 and 5D1). In the targeted (TG) allele, loss of the sequence between the homologous arms alters the distance between restriction enzyme sites, compared with the wild type (WT) allele. Consequently, in the wild type allele Probe A recognises a 5′ 8.7 kb fragment and Probe B a 3′ 10.8 kb fragment, whereas both probes detect a15.3 kb fragment for the targeted allele. C Western blot analysis of protein extracts from adult kidney and liver derived from Grb7 wild type (+ / +) and Grb7 KO (-/-) homozygous mice following establishment of a true breeding line. The blot was probed with an antibody specific for Grb7, that readily detects a species of approximately the correct size (65 kDa) in wild type but not Grb7 KO samples, whereas an antibody specific for α-tubulin (50 kDa) was detected equally in both

Techniques Used: Mutagenesis, Sequencing, Cloning, Homologous Recombination, Clone Assay, Plasmid Preparation, Construct, Selection, Stable Transfection, Virus, Southern Blot, Western Blot, Derivative Assay

Weights and blood glucose levels in PN1 progeny from Grb7 KO x Grb10 KO crosses. Weights of whole body and selected dissected organs, with blood glucose levels were collected at PN1 from progeny of crosses between Grb7 KO and Grb10 KO mice. Data were pooled into four groups for analysis as described in the Methods; wild type (WT), Grb7 KO (7KO), Grb10 KO (10KO) and Grb7 : Grb10 double knockout (DKO). For each of the four offspring genotype groups, data are shown for, A Body weight; and B Blood glucose concentration ([glu]). In addition, actual weights of ( C ) Brain; D Liver; E Lungs; F Heart and G Kidneys are shown above the relative weights of the same organs, expressed as a percentage of body mass ( H – L ). Values represent means and SEM, tested by one-way ANOVA using Kruskal–Wallis and Dunn’s post hoc statistical tests. Sample sizes were, for body and brain, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 15; kidneys and heart, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 48, Grb7 : Grb10 DKO N = 15, liver, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 14; lungs, WT N = 41, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 15; glucose levels, WT N = 24, Grb7 KO N = 7, Grb10 KO N = 27, Grb7 : Grb10 DKO N = 10. Asterisks indicate p- values, * p < 0.05, ** p < 0.01, **** p < 0.0001

Figure Legend Snippet: Weights and blood glucose levels in PN1 progeny from Grb7 KO x Grb10 KO crosses. Weights of whole body and selected dissected organs, with blood glucose levels were collected at PN1 from progeny of crosses between Grb7 KO and Grb10 KO mice. Data were pooled into four groups for analysis as described in the Methods; wild type (WT), Grb7 KO (7KO), Grb10 KO (10KO) and Grb7 : Grb10 double knockout (DKO). For each of the four offspring genotype groups, data are shown for, A Body weight; and B Blood glucose concentration ([glu]). In addition, actual weights of ( C ) Brain; D Liver; E Lungs; F Heart and G Kidneys are shown above the relative weights of the same organs, expressed as a percentage of body mass ( H – L ). Values represent means and SEM, tested by one-way ANOVA using Kruskal–Wallis and Dunn’s post hoc statistical tests. Sample sizes were, for body and brain, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 15; kidneys and heart, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 48, Grb7 : Grb10 DKO N = 15, liver, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 14; lungs, WT N = 41, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 15; glucose levels, WT N = 24, Grb7 KO N = 7, Grb10 KO N = 27, Grb7 : Grb10 DKO N = 10. Asterisks indicate p- values, * p < 0.05, ** p < 0.01, **** p < 0.0001

Techniques Used: Double Knockout, Concentration Assay

Weight analysis of e17.5 conceptuses from crosses between Grb7 KO and Grb10 KO mice. Data were pooled into four groups for analysis as described in the Methods; wild type (WT), Grb7 KO (7KO), Grb10 KO (10KO) and Grb7 : Grb10 double knockouts (DKO). Weights are shown for the four offspring genotype groups for ( A ) Embryo; and B Placenta. C These values have been used to calculate the embryo to placenta weight ratio as a measure of placental efficiency. Values represent means and SEM, tested by one-way ANOVA using Kruskal–Wallis and Dunn’s post hoc statistical tests. Sample sizes were, for embryo WT N = 24, Grb7 KO N = 5, Grb10 KO N = 27, Grb7 : Grb10 DKO N = 6, and for placenta and embryo:placenta ratio WT N = 23, Grb7 KO N = 5, Grb10 KO N = 27, Grb7:Grb10 DKO N = 6. Asterisks indicate p- values, * p < 0.05, ** p < 0.01, **** p < 0.0001

Figure Legend Snippet: Weight analysis of e17.5 conceptuses from crosses between Grb7 KO and Grb10 KO mice. Data were pooled into four groups for analysis as described in the Methods; wild type (WT), Grb7 KO (7KO), Grb10 KO (10KO) and Grb7 : Grb10 double knockouts (DKO). Weights are shown for the four offspring genotype groups for ( A ) Embryo; and B Placenta. C These values have been used to calculate the embryo to placenta weight ratio as a measure of placental efficiency. Values represent means and SEM, tested by one-way ANOVA using Kruskal–Wallis and Dunn’s post hoc statistical tests. Sample sizes were, for embryo WT N = 24, Grb7 KO N = 5, Grb10 KO N = 27, Grb7 : Grb10 DKO N = 6, and for placenta and embryo:placenta ratio WT N = 23, Grb7 KO N = 5, Grb10 KO N = 27, Grb7:Grb10 DKO N = 6. Asterisks indicate p- values, * p < 0.05, ** p < 0.01, **** p < 0.0001

Techniques Used:

Body composition analysis of adult Grb7 KO mice compared to wild type (WT) mice. Dual X-ray absorptiometry (DXA) analysis of 15 week old males ( A - F ) and females ( G - L ), showing estimates of total body weight ( A , G ), lean body content ( B , H ), fat body content ( C , I ), fat as a proportion of body weight ( D , J ), bone mineral content ( E , K ), and bone mineral density ( F , L ). For the same animals, physical weights were then obtained for the body and selected tissues and organs for both males ( M - R ) and females ( S - X ). Physical weight data are shown for total body ( M , S ), masseter muscle ( N , T ); gonadal WAT ( O , U ) and renal WAT ( Q , W ), along with weights as a proportion of body weight for gonadal WAT ( P , V ) and renal WAT ( R , X ). Graphs show means and SEM, and differences between the means were evaluated using a two-sided Student’s t-test. Sample sizes for DXA measurements were, for males WT N = 9, Grb7 KO N = 10 and females WT N = 5, Grb7 KO N = 3. Sample sizes for physical weight data were, for males WT N = 12, Grb7 KO N = 13, and for females WT N = 9, Grb7 KO N = 7. Asterisks indicate p- values, * p < 0.05

Figure Legend Snippet: Body composition analysis of adult Grb7 KO mice compared to wild type (WT) mice. Dual X-ray absorptiometry (DXA) analysis of 15 week old males ( A - F ) and females ( G - L ), showing estimates of total body weight ( A , G ), lean body content ( B , H ), fat body content ( C , I ), fat as a proportion of body weight ( D , J ), bone mineral content ( E , K ), and bone mineral density ( F , L ). For the same animals, physical weights were then obtained for the body and selected tissues and organs for both males ( M - R ) and females ( S - X ). Physical weight data are shown for total body ( M , S ), masseter muscle ( N , T ); gonadal WAT ( O , U ) and renal WAT ( Q , W ), along with weights as a proportion of body weight for gonadal WAT ( P , V ) and renal WAT ( R , X ). Graphs show means and SEM, and differences between the means were evaluated using a two-sided Student’s t-test. Sample sizes for DXA measurements were, for males WT N = 9, Grb7 KO N = 10 and females WT N = 5, Grb7 KO N = 3. Sample sizes for physical weight data were, for males WT N = 12, Grb7 KO N = 13, and for females WT N = 9, Grb7 KO N = 7. Asterisks indicate p- values, * p < 0.05

Techniques Used:

Glucose homeostasis in adult Grb7 KO mice compared to wild type. Circulating glucose levels were compared for both male ( A - C ) and female ( D - F ) wild type (WT) and Grb7 KO mice, aged 14 weeks, in the fasted ( A , D ) and fed ( B , E ) states. The same animals were also tested for the ability to clear a glucose load after a fasting period in a standard glucose tolerance test ( C , F ). Glucose levels were measured at intervals over a time-course of 120 min with areas under the curve (AUC) measured in each case for statistical comparison. Graphs show means and SEM, and differences between the means were evaluated using a two-sided Student’s t-test. Sample sizes for male fasted and fed glucose levels were WT N = 12, Grb7 KO N = 13, for female fasted glucose WT N = 9, Grb7 KO N = 7, and fed glucose WT N = 9, Grb7 KO N = 6. Sample sizes for glucose tolerance tests were, for males WT N = 10, Grb7 KO N = 11, and for females WT N = 9, Grb7 KO N = 7. Asterisks indicate p- values, * p < 0.05, ** p < 0.01

Figure Legend Snippet: Glucose homeostasis in adult Grb7 KO mice compared to wild type. Circulating glucose levels were compared for both male ( A - C ) and female ( D - F ) wild type (WT) and Grb7 KO mice, aged 14 weeks, in the fasted ( A , D ) and fed ( B , E ) states. The same animals were also tested for the ability to clear a glucose load after a fasting period in a standard glucose tolerance test ( C , F ). Glucose levels were measured at intervals over a time-course of 120 min with areas under the curve (AUC) measured in each case for statistical comparison. Graphs show means and SEM, and differences between the means were evaluated using a two-sided Student’s t-test. Sample sizes for male fasted and fed glucose levels were WT N = 12, Grb7 KO N = 13, for female fasted glucose WT N = 9, Grb7 KO N = 7, and fed glucose WT N = 9, Grb7 KO N = 6. Sample sizes for glucose tolerance tests were, for males WT N = 10, Grb7 KO N = 11, and for females WT N = 9, Grb7 KO N = 7. Asterisks indicate p- values, * p < 0.05, ** p < 0.01

Techniques Used: Comparison

Figure Legend Snippet: Grb7, Grb10 and Grb14 distribution in the developing mouse embryo

Techniques Used:

Image Search Results

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Grb7 expression in the mouse embryo visualised by immunohistochemistry on wild type e14.5 paraffin sections. Representative mid-sagittal sections were chosen to display a wide range of tissues (sections adjacent to those used to stain for Grb14 in Fig. ). A Whole wild type embryo with expression highlighted in dermis (d), gut (g), inner ear (i), liver (li), lung (lu), nasal epithelium (ne), pancreas (p), pituitary (pi), ribs (r, ossified cartilage), stomach (s), salivary gland (sg) and tooth primordia (tp); B Expression in submandibular gland and tooth primordia; C Expression in kidney (ki) and adrenal gland (ad); D Expression in lung and liver, but not heart (h); E Expression in epithelial lining of mid- and hind-gut; F Endocrine pancreas. Brown staining is indicative of Grb7 expression, magnifications as indicated

Article Snippet: For immunohistochemistry, protocols were essentially as previously described [ ], using primary antibodies specific for either Grb7 (Santa Cruz Biotechnology, CA, USA; GRB7 (N-20) N-terminal Grb7, rabbit polyclonal sc-607; RRID:AB_2113275), at 1:200 dilution, Grb10 (Santa Cruz Biotechnology, CA, USA; GRB10 (A-18) C-terminal α-Grb10, rabbit polyclonal sc-1027) at 1:200 dilution or Grb14 (Santa Cruz Biotechnology, CA, USA; GRB14 (N-19) N-terminal Grb14, goat polyclonal sc-6103; RRID:AB_2112989) at 1:100 dilution.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Grb14 expression in the mouse embryo visualised by immunohistochemistry on wild type e14.5 paraffin sections. Representative mid-sagittal sections were chosen to display a wide range of tissues (sections adjacent to those used to stain for Grb7 in Fig. ). A Whole wild type embryo with expression highlighted in cardiac muscle (c), choroid plexus (cp), dermis (d), diaphragm (di), gut (g), lungs (lu), liver (li), ribs (r, ossifying cartilage), stomach (s), tooth primordia (tp) and tongue (t); B Expression in choroid plexus ; C Expression in ossifying cartilage (ca) of vertebrae; D Expression in lung, ossifying cartilage of ribs and intercostal muscle (ic); E Expression in pancreas (p). Brown staining is indicative of Grb14 expression, magnifications as indicated

Techniques: Expressing, Immunohistochemistry, Staining

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Expression of Grb7 family members in PN21 mouse pancreas visualised by immunohistochemistry on paraffin sections. Antibodies specific for ( A ) Grb7; B Grb10; and C Grb14; were used to detect distinct proteins of the Grb7 family. Brown staining is indicative of protein expression. Arrows indicate endocrine area (pancreatic islets); magnification 100x

Techniques: Expressing, Immunohistochemistry, Staining

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Genetic crosses used in the study, showing parent and offspring genotypes with their expected ratios. Crosses between (A) Grb10 +/p : Grb14 +/- double heterozygous females and Grb10 +/p : Grb14 +/- double heterozygous males, producing offspring of twelve genotypes, and (B) Grb7 +/- : Grb10 +/p double heterozygous females and Grb7 +/- heterozygous males, producing offspring of six genotypes, each in the indicated, expected Mendelian ratios. For ANOVA statistical analysis these six or twelve genotypes were used to form four groups, as indicated, since no difference was anticipated between animals differing only in their Grb14 +/- and Grb14 +/+ or Grb7 +/- and Grb7 +/+ allelic status. Similarly, due to the imprinted expression of Grb10 , offspring inheriting a mutant copy of the paternal Grb10 allele ( Grb10 +/p ), having no growth phenotype, were not expected to differ from Grb10 wild type ( Grb10 +/+ ) offspring, while double knockout (DKO) offspring inheriting mutations of both parental alleles ( Grb10 m/p ) were expected to be indistinguishable from those inheriting a mutant copy of the normally active maternal Grb10 allele ( Grb10 m/+ )

Techniques: Expressing, Mutagenesis, Double Knockout

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Summary of PN1 body and organ weight data for progeny of crosses between (A) the Grb10 KO and Grb14 KO strains, and (B) the Grb7 KO and Grb10 KO strains. Mean weights are shown for each genotype together with changes relative to wild type (%WT) for each mutant genotype

Techniques: Mutagenesis

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Generation of a Grb7 KO mouse strain. A null mutation was designed, lacking all the protein coding sequence. A The wild type Grb7 locus (top), showing exons (filled boxes numbered 1–15), translational start (open triangle) and stop (filled triangle) codons, plus selected restriction enzyme sites. Note that the translational start (ATG) codon lies within a Nco I restriction site (CCATGG) used as part of the cloning strategy. Regions used to direct homologous recombination in ES cells (5’ arm and 3’ arm) and also probes used to confirm correct targeting are indicated to either side of the coding exons. The homologous arms were cloned into the pPNT vector to form the pPNT- Grb7 targeting construct (middle), in which the coding exons have been replaced with a neomycin ( neo ) resistance gene cassette for positive selection of ES cells stably incorporating the construct. The targeting construct also includes a herpes simplex virus thymidine kinase ( hsv-tk ) gene, located outside the homologous regions that is typically retained at sites of random integration, and was used to enrich for targeting events by negative selection. Consequently, the correctly targeted allele (bottom) retains the neo but not the hsv-tk gene. B Southern blot analysis of Hind III digested DNA from wild type ES cells (ES) or primary mouse embryonic fibroblasts (MEF) alongside three successfully targeted ES cell clones (2E5, 4B5 and 5D1). In the targeted (TG) allele, loss of the sequence between the homologous arms alters the distance between restriction enzyme sites, compared with the wild type (WT) allele. Consequently, in the wild type allele Probe A recognises a 5′ 8.7 kb fragment and Probe B a 3′ 10.8 kb fragment, whereas both probes detect a15.3 kb fragment for the targeted allele. C Western blot analysis of protein extracts from adult kidney and liver derived from Grb7 wild type (+ / +) and Grb7 KO (-/-) homozygous mice following establishment of a true breeding line. The blot was probed with an antibody specific for Grb7, that readily detects a species of approximately the correct size (65 kDa) in wild type but not Grb7 KO samples, whereas an antibody specific for α-tubulin (50 kDa) was detected equally in both

Techniques: Mutagenesis, Sequencing, Cloning, Homologous Recombination, Clone Assay, Plasmid Preparation, Construct, Selection, Stable Transfection, Virus, Southern Blot, Western Blot, Derivative Assay

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Weights and blood glucose levels in PN1 progeny from Grb7 KO x Grb10 KO crosses. Weights of whole body and selected dissected organs, with blood glucose levels were collected at PN1 from progeny of crosses between Grb7 KO and Grb10 KO mice. Data were pooled into four groups for analysis as described in the Methods; wild type (WT), Grb7 KO (7KO), Grb10 KO (10KO) and Grb7 : Grb10 double knockout (DKO). For each of the four offspring genotype groups, data are shown for, A Body weight; and B Blood glucose concentration ([glu]). In addition, actual weights of ( C ) Brain; D Liver; E Lungs; F Heart and G Kidneys are shown above the relative weights of the same organs, expressed as a percentage of body mass ( H – L ). Values represent means and SEM, tested by one-way ANOVA using Kruskal–Wallis and Dunn’s post hoc statistical tests. Sample sizes were, for body and brain, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 15; kidneys and heart, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 48, Grb7 : Grb10 DKO N = 15, liver, WT N = 42, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 14; lungs, WT N = 41, Grb7 KO N = 14, Grb10 KO N = 49, Grb7 : Grb10 DKO N = 15; glucose levels, WT N = 24, Grb7 KO N = 7, Grb10 KO N = 27, Grb7 : Grb10 DKO N = 10. Asterisks indicate p- values, * p < 0.05, ** p < 0.01, **** p < 0.0001

Techniques: Double Knockout, Concentration Assay

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Weight analysis of e17.5 conceptuses from crosses between Grb7 KO and Grb10 KO mice. Data were pooled into four groups for analysis as described in the Methods; wild type (WT), Grb7 KO (7KO), Grb10 KO (10KO) and Grb7 : Grb10 double knockouts (DKO). Weights are shown for the four offspring genotype groups for ( A ) Embryo; and B Placenta. C These values have been used to calculate the embryo to placenta weight ratio as a measure of placental efficiency. Values represent means and SEM, tested by one-way ANOVA using Kruskal–Wallis and Dunn’s post hoc statistical tests. Sample sizes were, for embryo WT N = 24, Grb7 KO N = 5, Grb10 KO N = 27, Grb7 : Grb10 DKO N = 6, and for placenta and embryo:placenta ratio WT N = 23, Grb7 KO N = 5, Grb10 KO N = 27, Grb7:Grb10 DKO N = 6. Asterisks indicate p- values, * p < 0.05, ** p < 0.01, **** p < 0.0001

Techniques:

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Body composition analysis of adult Grb7 KO mice compared to wild type (WT) mice. Dual X-ray absorptiometry (DXA) analysis of 15 week old males ( A - F ) and females ( G - L ), showing estimates of total body weight ( A , G ), lean body content ( B , H ), fat body content ( C , I ), fat as a proportion of body weight ( D , J ), bone mineral content ( E , K ), and bone mineral density ( F , L ). For the same animals, physical weights were then obtained for the body and selected tissues and organs for both males ( M - R ) and females ( S - X ). Physical weight data are shown for total body ( M , S ), masseter muscle ( N , T ); gonadal WAT ( O , U ) and renal WAT ( Q , W ), along with weights as a proportion of body weight for gonadal WAT ( P , V ) and renal WAT ( R , X ). Graphs show means and SEM, and differences between the means were evaluated using a two-sided Student’s t-test. Sample sizes for DXA measurements were, for males WT N = 9, Grb7 KO N = 10 and females WT N = 5, Grb7 KO N = 3. Sample sizes for physical weight data were, for males WT N = 12, Grb7 KO N = 13, and for females WT N = 9, Grb7 KO N = 7. Asterisks indicate p- values, * p < 0.05

Techniques:

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Glucose homeostasis in adult Grb7 KO mice compared to wild type. Circulating glucose levels were compared for both male ( A - C ) and female ( D - F ) wild type (WT) and Grb7 KO mice, aged 14 weeks, in the fasted ( A , D ) and fed ( B , E ) states. The same animals were also tested for the ability to clear a glucose load after a fasting period in a standard glucose tolerance test ( C , F ). Glucose levels were measured at intervals over a time-course of 120 min with areas under the curve (AUC) measured in each case for statistical comparison. Graphs show means and SEM, and differences between the means were evaluated using a two-sided Student’s t-test. Sample sizes for male fasted and fed glucose levels were WT N = 12, Grb7 KO N = 13, for female fasted glucose WT N = 9, Grb7 KO N = 7, and fed glucose WT N = 9, Grb7 KO N = 6. Sample sizes for glucose tolerance tests were, for males WT N = 10, Grb7 KO N = 11, and for females WT N = 9, Grb7 KO N = 7. Asterisks indicate p- values, * p < 0.05, ** p < 0.01

Techniques: Comparison

Journal: BMC Biology

doi: 10.1186/s12915-024-02018-5

Figure Lengend Snippet: Grb7, Grb10 and Grb14 distribution in the developing mouse embryo

Techniques:

A-C. A) Top: Domain topology of the Grb7 protein. The approximate locations of the Grb7 protein domains described in the text are identified by residue numbers located above the Grb7 schematic. The GM (Grbs and Mig) domain corresponds roughly to the homologous regions between the Grb7 and Mig 10 proteins. Bottom: Domain topology of the Filamin-a protein (dimer). The Filamin-a figure was adapted from Nakamura et al., 2011. B-C: β-galactosidase assays. B) The prey vector containing the IgFlna-16-19-AD (activation domain) was cotransformed in yeast with bait vectors containing the Grb7-RA-DBD (DNA binding domain), Grb7-PH-DBD, or Grb7-RA-PH-DBD, respectively (Figure shows the Grb7-RAPH-DBD experiment only). The p53/SV-40-T interaction serves as a positive control. C) β-galactosidase assay activity was determined by liquid assays using ONPG as substrate. The data represent averages with standard deviations from three independent experiments each done in triplicate.

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: A-C. A) Top: Domain topology of the Grb7 protein. The approximate locations of the Grb7 protein domains described in the text are identified by residue numbers located above the Grb7 schematic. The GM (Grbs and Mig) domain corresponds roughly to the homologous regions between the Grb7 and Mig 10 proteins. Bottom: Domain topology of the Filamin-a protein (dimer). The Filamin-a figure was adapted from Nakamura et al., 2011. B-C: β-galactosidase assays. B) The prey vector containing the IgFlna-16-19-AD (activation domain) was cotransformed in yeast with bait vectors containing the Grb7-RA-DBD (DNA binding domain), Grb7-PH-DBD, or Grb7-RA-PH-DBD, respectively (Figure shows the Grb7-RAPH-DBD experiment only). The p53/SV-40-T interaction serves as a positive control. C) β-galactosidase assay activity was determined by liquid assays using ONPG as substrate. The data represent averages with standard deviations from three independent experiments each done in triplicate.

Article Snippet: The following antibodies were obtained from Santa Cruz Biotechnology Inc, (Santa Cruz, CA): mouse monoclonal Filamin 1 (3 F180), mouse monoclonal Filamin 1 (PM6/317), rabbit polyclonal Grb7 (H-70), rabbit polyclonal Grb7 (C-20), mouse monoclonal GST (B-14), mouse monoclonal c-Myc (9E10), mouse monoclonal Actin (C-2), mouse monoclonal p-Tyr (PY20), goat anti-mouse IgG-HRP, and non-specific rabbit or mouse control IgG.

Techniques: Plasmid Preparation, Activation Assay, Binding Assay, Positive Control, Activity Assay

$A) GST binding assay: the IgFlna-16-19 domains bind with the Grb7-RA-PH domains. GST-IgFlna-16-19 and Grb7-RA-PH-6His were bacterially expressed and purified using glutathione (Glu-) sepharose beads and Ni-NTA agarose beads, respectively. Grb7-RA-PH-6His was incubated with GST-IgFlna-16-19 coupled glutathione beads and the eluted fraction was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained using Coomassie brilliant blue. Lanes: 1: protein standards, 2: purified GST-IgFlna-16-19, 3: Glu-sepharose beads elution fraction of GST-IgFlna-16-19 and Grb7-RA-PH-6His mixture, 4: purified Grb7-RA-PH-6His, 5: Glu-sepharose beads elution fraction of GST-IgFlna-16-19 and Grb7-RA-PH-6His mixture, 6: Glu-sepharose beads elution fraction of GST and Grb7-RA-PH-6His mixture, 7: Glu-sepharose beads elution fraction of Grb7-RAPH-6His alone. B) in vitro pull down of myc-Grb7-RA-PH by IgFlna-16-19. in vitro translated myc-tagged Grb7- RA-PH was incubated with purified glutathione coupled GST-IgFlna-16-19 (Lane 1) or GST alone (Lane 2). Lane 3 represents the elution fraction from glutathione coupled GST-IgFlna-16-19 only. Elution fractions were resolved by SDS-PAGE, and proteins were identified by Western blot analysis using mouse myc monoclonal antibody for Grb7-RA-PH detection and mouse GST monoclonal antibody for IgFlna-16-19 detection. C) In vivo coimmunoprecipitation of endogenous Grb7 and Filamin-a proteins. Left panels: coimmunoprecipitation with resin-coupled Grb7 antibody was performed on SKBR3 cell extracts endogenously expressing Grb7 and Filamin-a (Lanes 1–2, left upper panel). The whole cell lysate (WCL, lower left panel) and the resulting immunocomplexes (IP: Grb7) were immunoblotted with anti Grb7 (H-70), anti-Filamin-a (3 F-180), and anti-Actin (C-2) antibodies. A non-specific IgG control antibody immunoprecipitation (Lane 3, upper left panel) was also carried out on the extracts to show the interaction was specific. These experiments were also performed in MCF7 and Hela cells (Grb7 transfected), and similar results were observed (unpublished data). Right panels: the reverse process, that is, coimmunoprecipitation with resin-coupled Flna antibody was also performed (Lanes 1–2, upper right panel). The whole cell lysate (WCL, lower right panel), and the resulting immunocomplexes (IP: Filamin-a) were immunoblotted with Filamin-a and Grb7 antibodies. Non-specific IgG control and “non-antibody coupled beads” control coimmunoprecipitations (Lanes 3–4, upper right panel) show the interaction were specific. (COIP1: coimmunoprecipitation reaction1, COIP2: coimmunoprecipitation reaction 2, WCL1: whole cell lysate sample1, WCL2: whole cell lysate sample 2).$

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: A) GST binding assay: the IgFlna-16-19 domains bind with the Grb7-RA-PH domains. GST-IgFlna-16-19 and Grb7-RA-PH-6His were bacterially expressed and purified using glutathione (Glu-) sepharose beads and Ni-NTA agarose beads, respectively. Grb7-RA-PH-6His was incubated with GST-IgFlna-16-19 coupled glutathione beads and the eluted fraction was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained using Coomassie brilliant blue. Lanes: 1: protein standards, 2: purified GST-IgFlna-16-19, 3: Glu-sepharose beads elution fraction of GST-IgFlna-16-19 and Grb7-RA-PH-6His mixture, 4: purified Grb7-RA-PH-6His, 5: Glu-sepharose beads elution fraction of GST-IgFlna-16-19 and Grb7-RA-PH-6His mixture, 6: Glu-sepharose beads elution fraction of GST and Grb7-RA-PH-6His mixture, 7: Glu-sepharose beads elution fraction of Grb7-RAPH-6His alone. B) in vitro pull down of myc-Grb7-RA-PH by IgFlna-16-19. in vitro translated myc-tagged Grb7- RA-PH was incubated with purified glutathione coupled GST-IgFlna-16-19 (Lane 1) or GST alone (Lane 2). Lane 3 represents the elution fraction from glutathione coupled GST-IgFlna-16-19 only. Elution fractions were resolved by SDS-PAGE, and proteins were identified by Western blot analysis using mouse myc monoclonal antibody for Grb7-RA-PH detection and mouse GST monoclonal antibody for IgFlna-16-19 detection. C) In vivo coimmunoprecipitation of endogenous Grb7 and Filamin-a proteins. Left panels: coimmunoprecipitation with resin-coupled Grb7 antibody was performed on SKBR3 cell extracts endogenously expressing Grb7 and Filamin-a (Lanes 1–2, left upper panel). The whole cell lysate (WCL, lower left panel) and the resulting immunocomplexes (IP: Grb7) were immunoblotted with anti Grb7 (H-70), anti-Filamin-a (3 F-180), and anti-Actin (C-2) antibodies. A non-specific IgG control antibody immunoprecipitation (Lane 3, upper left panel) was also carried out on the extracts to show the interaction was specific. These experiments were also performed in MCF7 and Hela cells (Grb7 transfected), and similar results were observed (unpublished data). Right panels: the reverse process, that is, coimmunoprecipitation with resin-coupled Flna antibody was also performed (Lanes 1–2, upper right panel). The whole cell lysate (WCL, lower right panel), and the resulting immunocomplexes (IP: Filamin-a) were immunoblotted with Filamin-a and Grb7 antibodies. Non-specific IgG control and “non-antibody coupled beads” control coimmunoprecipitations (Lanes 3–4, upper right panel) show the interaction were specific. (COIP1: coimmunoprecipitation reaction1, COIP2: coimmunoprecipitation reaction 2, WCL1: whole cell lysate sample1, WCL2: whole cell lysate sample 2).

Techniques: Binding Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, In Vitro, Western Blot, In Vivo, Expressing, Immunoprecipitation, Transfection

The Grb10 RA-PH domain structure (Depetris et al., 2009, PDB# 3HK0). The left panel depicts the dimerized structure of the Grb10-RA-PH domains, with each RA and PH domains annotated. The right panel is an enlargement of the region outlined by dotted lines in the left panel. The Grb10 residues Y194 and Y341 (corresponding to Grb7 residues Y188 and Y338, respectively) are labeled for each RA-PH structure. The Grb10-RA-PH domain residues Q347 and N348, when mutated to alanine diminish dimerization, are marked by arrows.

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: The Grb10 RA-PH domain structure (Depetris et al., 2009, PDB# 3HK0). The left panel depicts the dimerized structure of the Grb10-RA-PH domains, with each RA and PH domains annotated. The right panel is an enlargement of the region outlined by dotted lines in the left panel. The Grb10 residues Y194 and Y341 (corresponding to Grb7 residues Y188 and Y338, respectively) are labeled for each RA-PH structure. The Grb10-RA-PH domain residues Q347 and N348, when mutated to alanine diminish dimerization, are marked by arrows.

Techniques: Labeling

The Filamin-a interaction with Grb7 tyrosine mutants is decreased. Upper panels: Western blot analysis. HeLa Cells were transfected with pCMV-FLGrb7- Y188F (full-length Grb7-Y188F mutant), pCMV-FLGrb7-Y338F, and pCMV-FLGrb7- Y188F/Y338F (double mutant) constructs. Cells were lysed and immunoprecipitated by resin-coupled rabbit polyclonal anti-Grb7 (H-70), followed by Western blot analysis with Filamin-a antibody (3 F-180) and Grb7 antibody (H-70). Whole cell lysates were also probed with the same antibodies and anti-Actin(c-2) antibody. The figure shown is a representative blot of three independent experiments, each displaying similar results. Lower panel: the results of three different immunoprecipitation experiments were analyzed by densitometry. Filamin-a band densities were divided by corresponding Grb7 mutant band densities and were shown to be normalized to Grb7 WT. Standard deviations were calculated for each sample data set and plotted in bar-graph format.

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: The Filamin-a interaction with Grb7 tyrosine mutants is decreased. Upper panels: Western blot analysis. HeLa Cells were transfected with pCMV-FLGrb7- Y188F (full-length Grb7-Y188F mutant), pCMV-FLGrb7-Y338F, and pCMV-FLGrb7- Y188F/Y338F (double mutant) constructs. Cells were lysed and immunoprecipitated by resin-coupled rabbit polyclonal anti-Grb7 (H-70), followed by Western blot analysis with Filamin-a antibody (3 F-180) and Grb7 antibody (H-70). Whole cell lysates were also probed with the same antibodies and anti-Actin(c-2) antibody. The figure shown is a representative blot of three independent experiments, each displaying similar results. Lower panel: the results of three different immunoprecipitation experiments were analyzed by densitometry. Filamin-a band densities were divided by corresponding Grb7 mutant band densities and were shown to be normalized to Grb7 WT. Standard deviations were calculated for each sample data set and plotted in bar-graph format.

Techniques: Western Blot, Transfection, Mutagenesis, Construct, Immunoprecipitation

Grb7 and Filamin-a colocalize within SKBR3 cell membrane ruffles. A) SKBR3 Cells grown on 18 mm cover slips were fixed and probed for Grb7 (red), Filamin-a (green), filamentous actin (cyan) and DNA (blue). Colocalization was observed on the membrane ruffles at the apical domain of cells (Panels A–D, arrowheads), whereas no colocalization was observed on the ventral or basal surface (Panels E–H). Representative fluorescent images of the cells are shown, and example colocalization of Grb7 and Filamin-a is indicated by an arrowhead. Bar 10 μm. B) EGF stimulation results in Grb7/ Filamin-a colocalization to cell membrane ruffles. Serum starved SKBR3 cells treated for 30 min with 100 ng/ml EGF resulted in profuse cell membrane ruffling (Panel A–D), whereas serum starved, unstimulated cells lacked any detectable colocalization. Bar 10 μm.

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: Grb7 and Filamin-a colocalize within SKBR3 cell membrane ruffles. A) SKBR3 Cells grown on 18 mm cover slips were fixed and probed for Grb7 (red), Filamin-a (green), filamentous actin (cyan) and DNA (blue). Colocalization was observed on the membrane ruffles at the apical domain of cells (Panels A–D, arrowheads), whereas no colocalization was observed on the ventral or basal surface (Panels E–H). Representative fluorescent images of the cells are shown, and example colocalization of Grb7 and Filamin-a is indicated by an arrowhead. Bar 10 μm. B) EGF stimulation results in Grb7/ Filamin-a colocalization to cell membrane ruffles. Serum starved SKBR3 cells treated for 30 min with 100 ng/ml EGF resulted in profuse cell membrane ruffling (Panel A–D), whereas serum starved, unstimulated cells lacked any detectable colocalization. Bar 10 μm.

Techniques:

A) Grb7 knockdown significantly decreases wound closure rate in a wound scratch assay. SKBR3 cells were transfected with Grb7shRNA or Scramble shRNA. A wound was made with a 10 μl pipette tip across each well of a 12-well plate, and images were captured at different time points (in hours) as indicated. The invasion of wound-space over time was assessed by counting the number of cells present within the original wound boundaries at respective time points. The effect of Grb7shRNA on wound closure was compared with that of scramble shRNA treated cells. B) Quantification of the comparative decrease in Grb7 expression in SKBR3 cells upon either transfection with Grb7-specific shRNA or scrambled shRNA is shown in both Western Blot and densitometric bar-graph format.

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: A) Grb7 knockdown significantly decreases wound closure rate in a wound scratch assay. SKBR3 cells were transfected with Grb7shRNA or Scramble shRNA. A wound was made with a 10 μl pipette tip across each well of a 12-well plate, and images were captured at different time points (in hours) as indicated. The invasion of wound-space over time was assessed by counting the number of cells present within the original wound boundaries at respective time points. The effect of Grb7shRNA on wound closure was compared with that of scramble shRNA treated cells. B) Quantification of the comparative decrease in Grb7 expression in SKBR3 cells upon either transfection with Grb7-specific shRNA or scrambled shRNA is shown in both Western Blot and densitometric bar-graph format.

Techniques: Wound Healing Assay, Transfection, shRNA, Transferring, Expressing, Western Blot

Review

Structured Review

Images

1) Product Images from "Grb7 , Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism"

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