rap1a c 17 Search Results


antibodies against rap1a  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology antibodies against rap1a
FIGURE 3. Inhibition of protein geranylgeranylation blocks HMGal ubiq- uitylation. UT-2/HMGal cells were incubated for 20 h in Medium B supple- mented with 50 M lovastatin. The cells were then treated with ZA and/or DGBP and/or GGTI-298, as indicated. Following a 1-h incubation, all dishes received 5 M MG-132 with or without sterols and/or MVA, as indicated, and were further incubated for 2 h. The cells were lysed in solution D, HMGal was immunoprecipitated (IP) with anti-HMGR membrane region antibodies and successively immunoblotted (IB) with mouse anti-ubiquitin and anti--galac- tosidase (-Gal) monoclonal antibodies. Aliquots of whole cell lysates (lower panel) were immunoblotted with an antibody that specifically recognizes unprenylated <t>Rap1a.</t>
Antibodies Against Rap1a, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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goat polyclonal ab against unprenylated rap 1a  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology goat polyclonal ab against unprenylated rap 1a
FIGURE 6. Expression of FPPS and Rap1 in monocytes and neutro- phils. Highly purified monocytes and neutrophils were incubated in me- dium alone or in the presence of 2, 10, or 50 mM of ZOL. After 3 h, the cells were washed with culture medium and incubated overnight in fresh medium. Cells were collected, washed with PBS, and lysed. Five micro- grams of cellular lysates were separated by SDS-PAGE. Proteins were transferred to nitrocellulose followed by immunoblot with anti-Rap1A/B mAb (clone 26B4) to detect total Rap1, with <t>polyclonal</t> Ab C-17 to detect <t>unprenylated</t> Rap1A (uRap1A), or anti-FPPS polyclonal Ab and appro- priate HRP-conjugated secondary Ab. Blots were stripped and GAPDH served as a loading control. Comparable results were obtained in a second independent experiment.
Goat Polyclonal Ab Against Unprenylated Rap 1a, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rap1a+c+17/SV40+T+Ag+Antibody/pm27619996-96-15-24
Average 96 stars, based on 1 article reviews
goat polyclonal ab against unprenylated rap 1a - by Bioz Stars, 2026-10
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rap1a c 17  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology rap1a c 17
FIGURE 2. Interaction between G, <t>Rap1a,</t> and Radil. A, Radil physically connects G and Rap1a. HEK293T cells were transfected with different plasmid combinations coding for Strep-HA-G2, G2, HA-Rap1a, and Venus-Radil. Strep-HA-G2-containing complexes were affinity-purified with streptavidin-Sepha- rose beads, and the association with Rap1a and Radil was monitored by Western blot (WB) with -HA and -GFP antibodies, respectively. The efficiency of G2 purification and expression was also followed by Western blot using HA antibodies. A fraction of the lysates for each samples was probed with -HA or -GFP antibodies to assess protein expression. The association of Rap1a with G2 was enhanced when Radil was overexpressed (compare lanes 2 and 3) (n 3). *, nonspecific band. B, formation of the GRap1aRadil complex requires active Rap1a. HEK293T cells were transfected with expression plasmids for Strep-HA- Radil and HA-Rap1a only (lane 2) or together with a vector coding for FLAG-Rap1GAP (lane 3). Strep-HA-Radil was purified using streptavidin-Sepharose beads, and its association with Rap1 and G was monitored by a Western blot using -HA and -G antibodies, respectively. The expression of Rap1GAP was followed using -FLAG antibodies. Although HA-Rap1a and G bound to Strep-HA-Radil (lane 2), these interactions were compromised in the presence of overex- pressed FLAG-Rap1GAP (lane 3) (n 4). C, expression of a constitutively active Rap1a mutant (Rap1aQ63E) promotes the interaction of G with Radil. Strep-HA-Radil expressing stable cells were transfected or not with HA-Rap1aQ63E. 48 h after transfection, Radil was affinity-purified with streptavidin- Sepharose beads for 1.5 h, and the eluates were analyzed by Western blot using the indicated antibodies. Untransfected HEK293T cells were used as the negative control for streptavidin purification. AP, affinity purification. Mr, molecular weights. K, 1000.
Rap1a C 17, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rap1a+c+17/IL-17+Antibody/10__1074_slash_jbc__m109__069948-45-16-18
Average 96 stars, based on 1 article reviews
rap1a c 17 - by Bioz Stars, 2026-10
96/100 stars
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Image Search Results


FIGURE 3. Inhibition of protein geranylgeranylation blocks HMGal ubiq- uitylation. UT-2/HMGal cells were incubated for 20 h in Medium B supple- mented with 50 M lovastatin. The cells were then treated with ZA and/or DGBP and/or GGTI-298, as indicated. Following a 1-h incubation, all dishes received 5 M MG-132 with or without sterols and/or MVA, as indicated, and were further incubated for 2 h. The cells were lysed in solution D, HMGal was immunoprecipitated (IP) with anti-HMGR membrane region antibodies and successively immunoblotted (IB) with mouse anti-ubiquitin and anti--galac- tosidase (-Gal) monoclonal antibodies. Aliquots of whole cell lysates (lower panel) were immunoblotted with an antibody that specifically recognizes unprenylated Rap1a.

Journal: Journal of Biological Chemistry

Article Title: Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

doi: 10.1074/jbc.m111.278036

Figure Lengend Snippet: FIGURE 3. Inhibition of protein geranylgeranylation blocks HMGal ubiq- uitylation. UT-2/HMGal cells were incubated for 20 h in Medium B supple- mented with 50 M lovastatin. The cells were then treated with ZA and/or DGBP and/or GGTI-298, as indicated. Following a 1-h incubation, all dishes received 5 M MG-132 with or without sterols and/or MVA, as indicated, and were further incubated for 2 h. The cells were lysed in solution D, HMGal was immunoprecipitated (IP) with anti-HMGR membrane region antibodies and successively immunoblotted (IB) with mouse anti-ubiquitin and anti--galac- tosidase (-Gal) monoclonal antibodies. Aliquots of whole cell lysates (lower panel) were immunoblotted with an antibody that specifically recognizes unprenylated Rap1a.

Article Snippet: Antibodies against Rap1A (c-17; SC-1482), Rap1 (c-121; SC-65), Rab6, (c-19; SC-310), and -actin (AC-15; SC-69879) were from Santa Cruz Biotechnology.

Techniques: Inhibition, Incubation, Immunoprecipitation, Membrane, Ubiquitin Proteomics, Bioprocessing

FIGURE 4. Dislocation of HMGal requires protein geranylgeranylation. A, UT-2/HMGal cells were incubated for 20 h in Medium B supplemented with 50 M lovastatin. The cells were then treated with ZA, DGBP, or both for 1 h before addition of 65 M ALLN, sterols, and/or MVA, as indicated. Following an additional 7-h incubation, the cells were permeabilized in Solution C and fractionated by centrifugation, as described under “Experimental Procedures.” Aliquots of the supernatant and pellet fractions were immunoblotted with the indicated antibodies. Unprenylated and total Rap1a was detected with goat (SC-1482) and rabbit (SC-65) antibodies, respectively. B, densitometric analysis of the blots. For each inhibitor, the -galactosidase immunoreactive signal in A, i (including trailing smear), was divided by the sum of -galactosidase immunoreactive signals in pellet and supernatant, setting the value as “100%” for cells not treated with either sterols or MVA. Results are the mean S.E. of 8 independent experiments. C, UT-2/HMGal cells were set up as in A. Cells were treated for 1 h with either 50 M GGTI-298 or 50 M FTI-277 before an additional 3-h incubation with 65 M ALLN and sterols, 2 mM MVA, or 20 mM MVA, as indicated. Cells were permeabilized and fractionated, as described. Aliquots of supernatant and pellet fractions were immunoblotted with the indicated antibodies. D, densitomet- ric analysis of the blots as in B, except treatment with 20 mM MVA was not quantified. Results are the mean S.E. of 3 independent experiments.

Journal: Journal of Biological Chemistry

Article Title: Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

doi: 10.1074/jbc.m111.278036

Figure Lengend Snippet: FIGURE 4. Dislocation of HMGal requires protein geranylgeranylation. A, UT-2/HMGal cells were incubated for 20 h in Medium B supplemented with 50 M lovastatin. The cells were then treated with ZA, DGBP, or both for 1 h before addition of 65 M ALLN, sterols, and/or MVA, as indicated. Following an additional 7-h incubation, the cells were permeabilized in Solution C and fractionated by centrifugation, as described under “Experimental Procedures.” Aliquots of the supernatant and pellet fractions were immunoblotted with the indicated antibodies. Unprenylated and total Rap1a was detected with goat (SC-1482) and rabbit (SC-65) antibodies, respectively. B, densitometric analysis of the blots. For each inhibitor, the -galactosidase immunoreactive signal in A, i (including trailing smear), was divided by the sum of -galactosidase immunoreactive signals in pellet and supernatant, setting the value as “100%” for cells not treated with either sterols or MVA. Results are the mean S.E. of 8 independent experiments. C, UT-2/HMGal cells were set up as in A. Cells were treated for 1 h with either 50 M GGTI-298 or 50 M FTI-277 before an additional 3-h incubation with 65 M ALLN and sterols, 2 mM MVA, or 20 mM MVA, as indicated. Cells were permeabilized and fractionated, as described. Aliquots of supernatant and pellet fractions were immunoblotted with the indicated antibodies. D, densitomet- ric analysis of the blots as in B, except treatment with 20 mM MVA was not quantified. Results are the mean S.E. of 3 independent experiments.

Article Snippet: Antibodies against Rap1A (c-17; SC-1482), Rap1 (c-121; SC-65), Rab6, (c-19; SC-310), and -actin (AC-15; SC-69879) were from Santa Cruz Biotechnology.

Techniques: Incubation, Centrifugation

FIGURE 8. Geranylgeranyl-PP synthesis is required for nonsterol-stimulated dislocation and degradation of HMGal. UT-2/HMGal cells were depleted of sterols and MVA by 20 h incubation in Medium B containing 50 M lovastatin. The cells were treated with 250 M ZOL, 150 M ZA, 10 M NB-598, 5 M RO-48-8071, or 10 M SKF-104976 with or without 250 M DGBP for 1 h before adding 65 M ALLN and 20 mM MVA, as indicated. Following an additional 7-h incubation, the cells were permeabilized and fractionated. A, aliquots of supernatant and solubilized pellet fraction were immunoblotted (IB) with anti-- galactosidase and anti-unprenylated Rap1a antibodies. B, densitometric analysis of the blots. The -galactosidase immunoreactive signal in supernatants in upper panel A (including trailing smear) was divided by the sum of -galactosidase immunoreactive signal in supernatant and pellet, setting the value for untreated cells as “100% dislocation.” Black bars, cells not treated with DGBP; gray bars, DGBP-treated cells. Results are the mean S.E. of 3 independent experiments. C, UT-2/HMGal cells were set up as in A. The cells were pulse-labeled and chased for 6 h in Medium B supplemented without (lane 2) or with 20 mM MVA (lanes 3–14) in the absence or presence of DGBP and the indicated inhibitors: 250 M ZOL, 150 M ZA, 10 M NB-598, 5 M RO-48–8071, or 10 M SKF-104976. (i) HMGal was immunoprecipitated (IP) with anti-HMGR membrane region polyclonal antibodies. The remaining HMGal at the end of the chase (%) is indicated. (ii) Aliquots of the labeled cell lysates were immunoblotted with the indicated antibodies.

Journal: Journal of Biological Chemistry

Article Title: Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

doi: 10.1074/jbc.m111.278036

Figure Lengend Snippet: FIGURE 8. Geranylgeranyl-PP synthesis is required for nonsterol-stimulated dislocation and degradation of HMGal. UT-2/HMGal cells were depleted of sterols and MVA by 20 h incubation in Medium B containing 50 M lovastatin. The cells were treated with 250 M ZOL, 150 M ZA, 10 M NB-598, 5 M RO-48-8071, or 10 M SKF-104976 with or without 250 M DGBP for 1 h before adding 65 M ALLN and 20 mM MVA, as indicated. Following an additional 7-h incubation, the cells were permeabilized and fractionated. A, aliquots of supernatant and solubilized pellet fraction were immunoblotted (IB) with anti-- galactosidase and anti-unprenylated Rap1a antibodies. B, densitometric analysis of the blots. The -galactosidase immunoreactive signal in supernatants in upper panel A (including trailing smear) was divided by the sum of -galactosidase immunoreactive signal in supernatant and pellet, setting the value for untreated cells as “100% dislocation.” Black bars, cells not treated with DGBP; gray bars, DGBP-treated cells. Results are the mean S.E. of 3 independent experiments. C, UT-2/HMGal cells were set up as in A. The cells were pulse-labeled and chased for 6 h in Medium B supplemented without (lane 2) or with 20 mM MVA (lanes 3–14) in the absence or presence of DGBP and the indicated inhibitors: 250 M ZOL, 150 M ZA, 10 M NB-598, 5 M RO-48–8071, or 10 M SKF-104976. (i) HMGal was immunoprecipitated (IP) with anti-HMGR membrane region polyclonal antibodies. The remaining HMGal at the end of the chase (%) is indicated. (ii) Aliquots of the labeled cell lysates were immunoblotted with the indicated antibodies.

Article Snippet: Antibodies against Rap1A (c-17; SC-1482), Rap1 (c-121; SC-65), Rab6, (c-19; SC-310), and -actin (AC-15; SC-69879) were from Santa Cruz Biotechnology.

Techniques: Incubation, Labeling, Immunoprecipitation, Membrane

FIGURE 6. Expression of FPPS and Rap1 in monocytes and neutro- phils. Highly purified monocytes and neutrophils were incubated in me- dium alone or in the presence of 2, 10, or 50 mM of ZOL. After 3 h, the cells were washed with culture medium and incubated overnight in fresh medium. Cells were collected, washed with PBS, and lysed. Five micro- grams of cellular lysates were separated by SDS-PAGE. Proteins were transferred to nitrocellulose followed by immunoblot with anti-Rap1A/B mAb (clone 26B4) to detect total Rap1, with polyclonal Ab C-17 to detect unprenylated Rap1A (uRap1A), or anti-FPPS polyclonal Ab and appro- priate HRP-conjugated secondary Ab. Blots were stripped and GAPDH served as a loading control. Comparable results were obtained in a second independent experiment.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

doi: 10.4049/jimmunol.1600913

Figure Lengend Snippet: FIGURE 6. Expression of FPPS and Rap1 in monocytes and neutro- phils. Highly purified monocytes and neutrophils were incubated in me- dium alone or in the presence of 2, 10, or 50 mM of ZOL. After 3 h, the cells were washed with culture medium and incubated overnight in fresh medium. Cells were collected, washed with PBS, and lysed. Five micro- grams of cellular lysates were separated by SDS-PAGE. Proteins were transferred to nitrocellulose followed by immunoblot with anti-Rap1A/B mAb (clone 26B4) to detect total Rap1, with polyclonal Ab C-17 to detect unprenylated Rap1A (uRap1A), or anti-FPPS polyclonal Ab and appro- priate HRP-conjugated secondary Ab. Blots were stripped and GAPDH served as a loading control. Comparable results were obtained in a second independent experiment.

Article Snippet: Western blots were separately analyzed using rabbit polyclonal Ab against FPPS (PA5-28228; Thermo Fisher Scientific), goat polyclonal Ab against unprenylated Rap 1A (C-17, sc-1482; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit mAb against total Rap1 (2399; Cell Signaling Technology, Danvers, MA) and processed as described previously (37) with corresponding HRP-coupled secondary Ab and chemiluminescence reagents from GE Healthcare (Freiburg, Germany).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Control

FIGURE 2. Interaction between G, Rap1a, and Radil. A, Radil physically connects G and Rap1a. HEK293T cells were transfected with different plasmid combinations coding for Strep-HA-G2, G2, HA-Rap1a, and Venus-Radil. Strep-HA-G2-containing complexes were affinity-purified with streptavidin-Sepha- rose beads, and the association with Rap1a and Radil was monitored by Western blot (WB) with -HA and -GFP antibodies, respectively. The efficiency of G2 purification and expression was also followed by Western blot using HA antibodies. A fraction of the lysates for each samples was probed with -HA or -GFP antibodies to assess protein expression. The association of Rap1a with G2 was enhanced when Radil was overexpressed (compare lanes 2 and 3) (n 3). *, nonspecific band. B, formation of the GRap1aRadil complex requires active Rap1a. HEK293T cells were transfected with expression plasmids for Strep-HA- Radil and HA-Rap1a only (lane 2) or together with a vector coding for FLAG-Rap1GAP (lane 3). Strep-HA-Radil was purified using streptavidin-Sepharose beads, and its association with Rap1 and G was monitored by a Western blot using -HA and -G antibodies, respectively. The expression of Rap1GAP was followed using -FLAG antibodies. Although HA-Rap1a and G bound to Strep-HA-Radil (lane 2), these interactions were compromised in the presence of overex- pressed FLAG-Rap1GAP (lane 3) (n 4). C, expression of a constitutively active Rap1a mutant (Rap1aQ63E) promotes the interaction of G with Radil. Strep-HA-Radil expressing stable cells were transfected or not with HA-Rap1aQ63E. 48 h after transfection, Radil was affinity-purified with streptavidin- Sepharose beads for 1.5 h, and the eluates were analyzed by Western blot using the indicated antibodies. Untransfected HEK293T cells were used as the negative control for streptavidin purification. AP, affinity purification. Mr, molecular weights. K, 1000.

Journal: Journal of Biological Chemistry

Article Title: G Protein βγ Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.m109.069948

Figure Lengend Snippet: FIGURE 2. Interaction between G, Rap1a, and Radil. A, Radil physically connects G and Rap1a. HEK293T cells were transfected with different plasmid combinations coding for Strep-HA-G2, G2, HA-Rap1a, and Venus-Radil. Strep-HA-G2-containing complexes were affinity-purified with streptavidin-Sepha- rose beads, and the association with Rap1a and Radil was monitored by Western blot (WB) with -HA and -GFP antibodies, respectively. The efficiency of G2 purification and expression was also followed by Western blot using HA antibodies. A fraction of the lysates for each samples was probed with -HA or -GFP antibodies to assess protein expression. The association of Rap1a with G2 was enhanced when Radil was overexpressed (compare lanes 2 and 3) (n 3). *, nonspecific band. B, formation of the GRap1aRadil complex requires active Rap1a. HEK293T cells were transfected with expression plasmids for Strep-HA- Radil and HA-Rap1a only (lane 2) or together with a vector coding for FLAG-Rap1GAP (lane 3). Strep-HA-Radil was purified using streptavidin-Sepharose beads, and its association with Rap1 and G was monitored by a Western blot using -HA and -G antibodies, respectively. The expression of Rap1GAP was followed using -FLAG antibodies. Although HA-Rap1a and G bound to Strep-HA-Radil (lane 2), these interactions were compromised in the presence of overex- pressed FLAG-Rap1GAP (lane 3) (n 4). C, expression of a constitutively active Rap1a mutant (Rap1aQ63E) promotes the interaction of G with Radil. Strep-HA-Radil expressing stable cells were transfected or not with HA-Rap1aQ63E. 48 h after transfection, Radil was affinity-purified with streptavidin- Sepharose beads for 1.5 h, and the eluates were analyzed by Western blot using the indicated antibodies. Untransfected HEK293T cells were used as the negative control for streptavidin purification. AP, affinity purification. Mr, molecular weights. K, 1000.

Article Snippet: Antibodies were purchased from the following vendors: -HA.11 (Covance), -FLAG (Sigma), -G (T20) (Santa Cruz Biotechnology), -Rap1a (C-17) (Santa Cruz Biotechnology), -Erk1/2 (Cell Signaling), -CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), -GFP (Covance and the University of Alberta), -tubulin (Sigma), and -Na-K-ATPase (Affinity Bioreagents).

Techniques: Transfection, Plasmid Preparation, Affinity Purification, Western Blot, Purification, Expressing, Mutagenesis, Negative Control

FIGURE 3. Rap1a-GTP and G promote the translocation of Radil at cell-matrix contacts in HT1080 cells. A, shown is localization of GFP (a and b), Venus-Radil (c and d), or Venus-Radil co-expressed with wild-type HA-Rap1 (e–g), HA-Rap1aQ63E (h–j), HA-Rap1G12V (k–m), or FLAG-G2/G1 (n–p). HA- and FLAG-tagged Rap1a or G2 G (untagged) were transiently transfected in HT1080 cells stably expressing Venus-Radil as indicated. Cells were fixed followed by immunodetection with -HA (e–m) or -FLAG (n–p) monoclonal antibody followed by secondary detection with goat-anti-mouse conjugated to Alexa 594 antibody. Cells were visualized using a Zeiss LSM 510 confocal microscope under 63 oil immersion objective. White arrows depict plasma-membrane co-localization. White boxes indicate magnified sections cropped in Adobe Photoshop CS3. Images shown are representative of 10–15 cells analyzed in three independent experiments. Bars, 50 m. B, membrane fractions show enrichment of Venus-Radil when Rap1aQ63E and G are expressed. HT1080 cells stably expressing Venus-Radil were transiently transfected to express the indicated proteins. Cells were lysed in hypotonic buffer and subjected to subcellular fractionationasdescribedunder“ExperimentalProcedures.”MembranefractionsandtheinputswereanalyzedbyWesternblot(WB)todeterminetheamount of Venus-Radil present in the membrane pool when HA-Rap1aQ63E or FLAG-G2 was co-expressed. The blot for the membrane fractions was stripped and re-probed using -Na-K-ATPase antibody to provide for an internal loading control.

Journal: Journal of Biological Chemistry

Article Title: G Protein βγ Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.m109.069948

Figure Lengend Snippet: FIGURE 3. Rap1a-GTP and G promote the translocation of Radil at cell-matrix contacts in HT1080 cells. A, shown is localization of GFP (a and b), Venus-Radil (c and d), or Venus-Radil co-expressed with wild-type HA-Rap1 (e–g), HA-Rap1aQ63E (h–j), HA-Rap1G12V (k–m), or FLAG-G2/G1 (n–p). HA- and FLAG-tagged Rap1a or G2 G (untagged) were transiently transfected in HT1080 cells stably expressing Venus-Radil as indicated. Cells were fixed followed by immunodetection with -HA (e–m) or -FLAG (n–p) monoclonal antibody followed by secondary detection with goat-anti-mouse conjugated to Alexa 594 antibody. Cells were visualized using a Zeiss LSM 510 confocal microscope under 63 oil immersion objective. White arrows depict plasma-membrane co-localization. White boxes indicate magnified sections cropped in Adobe Photoshop CS3. Images shown are representative of 10–15 cells analyzed in three independent experiments. Bars, 50 m. B, membrane fractions show enrichment of Venus-Radil when Rap1aQ63E and G are expressed. HT1080 cells stably expressing Venus-Radil were transiently transfected to express the indicated proteins. Cells were lysed in hypotonic buffer and subjected to subcellular fractionationasdescribedunder“ExperimentalProcedures.”MembranefractionsandtheinputswereanalyzedbyWesternblot(WB)todeterminetheamount of Venus-Radil present in the membrane pool when HA-Rap1aQ63E or FLAG-G2 was co-expressed. The blot for the membrane fractions was stripped and re-probed using -Na-K-ATPase antibody to provide for an internal loading control.

Article Snippet: Antibodies were purchased from the following vendors: -HA.11 (Covance), -FLAG (Sigma), -G (T20) (Santa Cruz Biotechnology), -Rap1a (C-17) (Santa Cruz Biotechnology), -Erk1/2 (Cell Signaling), -CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), -GFP (Covance and the University of Alberta), -tubulin (Sigma), and -Na-K-ATPase (Affinity Bioreagents).

Techniques: Translocation Assay, Transfection, Stable Transfection, Expressing, Immunodetection, Microscopy, Clinical Proteomics, Membrane, Control

FIGURE 5. Radil and G promote adhesion of HT1080 cells on fibronectin matrix in a Rap1a dependent manner. A, shown are representative pictures of HT1080 cell adhesion assays in response to overexpression of different proteins as indicated. FLAG-GFP cDNA was transfected as control. Top panels show the remaining adherent cells after washes. The bottom panels demonstrate the total cells input for each conditions. B, shown is a quantified representation of the adhesion assay as -fold stimulation compared with control. C, shown are representative pictures comparing the ability of RA and PDZ Radil mutants to promote HT1080 cells adhesion. D, shown is a quantified representation of C. The inset shows expression of the different Radil proteins in one representative experiment. Each experiment was done in triplicate, and several pictures from random fields were taken from each well of 96-well plate. Cells were counted (using an automated cell counting macro created in ImageJ software) from each field and expressed as -fold of control from each experiment. The results from three independent experiments were pooled for the quantification. Each dot on the graph represents cell counts from a single field. Error bars, S.E. Statistics are one-way ANOVA followed by Tukey’s multiple comparison test; p 0.05. Asterisks indicate statistical significance compared with control.

Journal: Journal of Biological Chemistry

Article Title: G Protein βγ Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.m109.069948

Figure Lengend Snippet: FIGURE 5. Radil and G promote adhesion of HT1080 cells on fibronectin matrix in a Rap1a dependent manner. A, shown are representative pictures of HT1080 cell adhesion assays in response to overexpression of different proteins as indicated. FLAG-GFP cDNA was transfected as control. Top panels show the remaining adherent cells after washes. The bottom panels demonstrate the total cells input for each conditions. B, shown is a quantified representation of the adhesion assay as -fold stimulation compared with control. C, shown are representative pictures comparing the ability of RA and PDZ Radil mutants to promote HT1080 cells adhesion. D, shown is a quantified representation of C. The inset shows expression of the different Radil proteins in one representative experiment. Each experiment was done in triplicate, and several pictures from random fields were taken from each well of 96-well plate. Cells were counted (using an automated cell counting macro created in ImageJ software) from each field and expressed as -fold of control from each experiment. The results from three independent experiments were pooled for the quantification. Each dot on the graph represents cell counts from a single field. Error bars, S.E. Statistics are one-way ANOVA followed by Tukey’s multiple comparison test; p 0.05. Asterisks indicate statistical significance compared with control.

Article Snippet: Antibodies were purchased from the following vendors: -HA.11 (Covance), -FLAG (Sigma), -G (T20) (Santa Cruz Biotechnology), -Rap1a (C-17) (Santa Cruz Biotechnology), -Erk1/2 (Cell Signaling), -CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), -GFP (Covance and the University of Alberta), -tubulin (Sigma), and -Na-K-ATPase (Affinity Bioreagents).

Techniques: Over Expression, Transfection, Control, Cell Adhesion Assay, Expressing, Cell Counting, Software, Comparison

FIGURE 6. fMLP promotes Rap1-dependent HT1080 cell adhesion, Rap1 activation, and Radil translocation to the plasma membrane. A, HT1080 cell adhesion on a fibronectin matrix is shown with or without fMLP (100 nM) stimulation in the presence or absence of Rap1GAP. Rap1aQ63E (used as a positive control) shows the increase in cell adhesion (n 3). B, shown is the time course of Rap1 activation by fMLP (100 nM) in HT1080 cells. The top panel shows GTP-bound Rap1a, purified using GST-RalGDS-RBD pulldown assays. The bottom panel shows total Rap1 in a fraction of whole cell lysates from each sample. Western blotting (WB) was done using -Rap1a antibodies. C, HT1080 cells stably expressing Venus-Radil were serum-starved for 24 h and stimulated or not with fMLP (1 M) for 0 min (a), 2 min (b), 3 min (c) at 37 °C. Arrows highlight regions of Venus-Radil enrichment at the plasma membrane. Images were captured by a blinded observer using 100 oil-immersion lens. Images shown are representative of two independent experiments. Bars, 20 m.

Journal: Journal of Biological Chemistry

Article Title: G Protein βγ Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.m109.069948

Figure Lengend Snippet: FIGURE 6. fMLP promotes Rap1-dependent HT1080 cell adhesion, Rap1 activation, and Radil translocation to the plasma membrane. A, HT1080 cell adhesion on a fibronectin matrix is shown with or without fMLP (100 nM) stimulation in the presence or absence of Rap1GAP. Rap1aQ63E (used as a positive control) shows the increase in cell adhesion (n 3). B, shown is the time course of Rap1 activation by fMLP (100 nM) in HT1080 cells. The top panel shows GTP-bound Rap1a, purified using GST-RalGDS-RBD pulldown assays. The bottom panel shows total Rap1 in a fraction of whole cell lysates from each sample. Western blotting (WB) was done using -Rap1a antibodies. C, HT1080 cells stably expressing Venus-Radil were serum-starved for 24 h and stimulated or not with fMLP (1 M) for 0 min (a), 2 min (b), 3 min (c) at 37 °C. Arrows highlight regions of Venus-Radil enrichment at the plasma membrane. Images were captured by a blinded observer using 100 oil-immersion lens. Images shown are representative of two independent experiments. Bars, 20 m.

Article Snippet: Antibodies were purchased from the following vendors: -HA.11 (Covance), -FLAG (Sigma), -G (T20) (Santa Cruz Biotechnology), -Rap1a (C-17) (Santa Cruz Biotechnology), -Erk1/2 (Cell Signaling), -CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), -GFP (Covance and the University of Alberta), -tubulin (Sigma), and -Na-K-ATPase (Affinity Bioreagents).

Techniques: Activation Assay, Translocation Assay, Clinical Proteomics, Membrane, Positive Control, Purification, Western Blot, Stable Transfection, Expressing