mouse anti grp75 antibody ab  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: a SKOV-3 cells were incubated with scFv-αHSM nanoparticles at 37 °C for 1 h uptake. Endocytic vesicles were isolated and purified by the magnetic pick-pen method as described in “Materials and methods” (Supplementary Fig. ). The PNS (post-nuclear supernatant. cell nuclei, and debris were removed), Sup (the supernatant was washed out during magnetic purification) and Pel (magnetic separation of vesicular pellets) fractions from cells pre-treated with/without MG132 (50 μM, 6 h) were subjected to SDS-PAGE, and vesicular proteins were analyzed by Western blot using the indicated Abs. b MG132-pre-treated SKOV-3 cells were incubated for 1 h at 37 °C with scFv-αHS-AF647 complexes, and the endogenous GRP75 and Dbl were stained by primary Abs followed by the AF488-conjugated secondary Ab. The co-localization of scFv-αHS complexes with indicated proteins was visualized using confocal microscopy. c , d , e , f Dbl knockdown SKOV-3 stable cell lines (Dbl-KD; D1, D2, D3) were produced by the CRISPR–CAS9 system. The uptakes of Tfn-AF647, Dextran-Rhodamine, scFv-αHS-AF647, and Tat/pGL3-YOYO-1 complexes in untransfected SKOV-3 cells (Ctrl, set as 100%), in Dbl-KD (D3) cell populations, or in proto- or onco-Dbl overexpression-rescued cell populations were immediately quantified by FACS. 10,000 cells were counted per sample with triplicate samples per transfection for each experiment, n = 3. Statistically significant differences compared with the uptakes in Scramble-sgRNA stable cell lines (NC) are shown: ** P < 0.01, * P < 0.05

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques: Incubation, Isolation, Purification, SDS Page, Western Blot, Staining, Confocal Microscopy, Knockdown, Stable Transfection, Produced, CRISPR, Over Expression, Transfection

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: a Western blot determined the steady-state level of endogenous Dbl in SKOV-3 cells (lane 1) and exogenously expressed GST-proto-Dbl (lane 2) or GST-onco-Dbl (lane 3) in transfected Cos-7 cells; c SKOV-3 cells were treated with MG132 (6 h) at indicated concentrations for inhibition of the ubiquitin–proteasome pathway. The degradation of proto-Dbl was analyzed by Western blot; Representative blot results from three independent experiments are shown in a , c . Protein brands were quantified by Image J software, and the expression ratio or variation of Dbl proteins were quantified and are shown in b , e SKOV-3 cells and transfected Cos-7 cells were stained by anti-GRP75 Ab (anti-rabbit AF488 as 2nd Ab) together with anti-Dbl Ab (or anti-GST Ab and anti-mouse AF647 as 2nd Ab). The subcellular co-localization of GR75 with endogenous Dbl or with exogenous GST-proto-Dbl/onco-Dbl was analyzed by confocal imaging. Endogenous Dbl and GRP75 were respectively co-immunoprecipitated (co-IP) by mouse anti-GRP75 Ab (lane 2) ( f ) and rabbit anti-Dbl Ab (lane 2) ( g ) in SKOV-3 cells. Proteins precipitates together with cell lysates (lane 1) were subjected to Western blot analysis using the indicated Abs. The isotype-matched IgG (lane 3) was used as the control. The asterisk points to an unspecific band when blotting with anti-Dbl Abs (proto-Dbl, 103 kDa, and onco-Dbl, 47 kDa); h Cos-7 cells were transfected with different Dbl constructs (as described in “Materials and methods”) followed by MG132 (25 μM, 6 h) treatment (DMSO as control). Cells were lysed 48 h post-transfection, and the lysates were subjected to GST pull-down. The precipitates were resolved by SDS-PAGE, and the co-pull-down protein was blotted using the indicated Abs. GST-P: pEBG-GST plasmid. GST-N-Dbl: N-terminal cloned Dbl (residues 1–498) into the pEBG-GST plasmid

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques: Western Blot, Transfection, Inhibition, Ubiquitin Proteomics, Software, Expressing, Staining, Imaging, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Construct, SDS Page, Plasmid Preparation, Clone Assay

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: a Dbl-KD (D3) and NC SKOV-3 stable cell lines were transfected either with the GRP75-targeting shRNA Lentivirus (GRP75-KD), or with GRP75-pEGFP plasmids, or further treated with its chemical inhibitor MKT077 (40 µM, 12 h). After culture for 36 h, cells had Tfn-AF647, CTxB-AF647, Dextran-Rhodamine, scFv-αHS-AF647, and Tat/pGL3-YOYO-1 complexes added for 37 °C uptake as described in “Materials and methods”. The uptakes of each drug were determined by confocal imaging analysis, and representative images from three independent experiments are shown. Scale bar: 20 µm. The uptake variability of indicated transfections/treatments in single cell populations is shown in Supplementary Fig. ; b GRP75 knockdown Cos-7 stable cell lines (GRP75-KD; G1, G2, G3) were produced by the CRISPR–CAS9 system. GRP75-KD (G3), NC Cos-7 stable cell lines, and MKT077-treated Cos-7 cells were transfected with either GST-proto-Dbl or GST-onco-Dbl plasmids. The incubation of fluorescent-labeled drugs and confocal-based uptake analysis were as described as above. The uptake variability in single cell populations is shown in Supplementary Fig.

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques: Stable Transfection, Transfection, shRNA, Imaging, Knockdown, Produced, CRISPR, Incubation, Labeling

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: a GRP75 knockdown stable cell lines (GRP75-KD: G1, G2, G3) were produced from SKOV-3 cells (WT) by the CRISPR–CAS9 system. pLenti-CRISPR v2 plasmid transfected SKOV-3 cells was set as the control (NC). The protein levels of endogenous proto-Dbl, onco-Dbl and GRP75 were determined by Western blot using the indicated Abs. c SKOV-3 cells were treated with/without CHX (100 µg/ml) for 12 h to inhibit de novo protein synthesis, and the GRP75 inhibitor MKT077 was added either in a time-dependent or in a concentration-dependent manner. The protein levels of endogenous proto- and onco-Dbl were analyzed by Western blot. e GRP75-KD stable cell lines (G2, G3) were pre-treated with the UPP inhibitor MG132 (50 μM, 6 h) before harvest. DMSO treatment was used as the control. The protein level changes of proto- and onco-Dbl after MG132 treatment was analyzed by Western blot. Representative blot results from three independent experiments are shown in a , c , e , and corresponding quantitation data (determined similarly as above) are shown in b , d , f . Statistically significant differences compared with corresponding NC groups are shown: ** P < 0.01

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques: Knockdown, Stable Transfection, Produced, CRISPR, Plasmid Preparation, Transfection, Control, Western Blot, Concentration Assay, Quantitation Assay

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: a Endogenous Dbl was immunoprecipitated by rabbit anti-Dbl Ab from the lysate of SKOV-3 cells pre-treated with/without MG132 (50 μM, 6 h). Isotype-matched rabbit IgG was used as the control. Dbl and ubiquitinated proto-Dbl from the precipitates were determined by Western blot using rabbit anti-Dbl Ab and mouse anti-ubiquitin (Ub) Ab, respectively. b Cos-7 cells were first co-transfected with GST-Dbl constructs and HA-Ub plasmids for 40 h culture, then treated with MG132 (25 μM) for 6 h. Exogenous Dbl was pull-down from cell lysate by glutathione sepharose 4B, ectopically expressed and ubiquitinated GST-Dbl was determined by Western blot using mouse anti-GST Ab and mouse anti-HA Ab, respectively. c GRP75-KD (G3) and NC SKOV-3 stable cell lines were treated with MG132 before harvest. After immunoprecipitation by rabbit anti-Dbl Ab, the Dbl protein level, and its ubiquitin modification were evaluated by Western blot using rabbit anti-Dbl Ab and mouse anti-Ub Ab, respectively. The isotype-matched IgG was used as the control; d GRP75-KD (G3) and NC Cos-7 stable cell lines were co-transfected with GST-Dbl constructs and HA-Ub plasmids followed by MG132 treatment as described above. The ectopic expressed GST-Dbl proteins and their ubiquitination changes were analyzed by Western blot using rabbit anti-GST Ab and mouse anti-Ub Ab, respectively. Representative blot results from three independent experiments are shown

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques: Immunoprecipitation, Control, Western Blot, Ubiquitin Proteomics, Transfection, Construct, Stable Transfection, Modification

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: a GRP75 KD (G3) and NC Cos-7 stable cell lines were co-transfected with GST-Dbl constructs and myc-CHIP plasmids. Exogenous GST-proto-Dbl were pull-down from cell lysate by mouse anti-GST Ab, and co-pull-down CHIP, Hsc70, and HSP90 were evaluated by their corresponding Ab, respectively. b GRP75 KD (G3) Cos-7 stable cells were co-transfected with GST-Dbl constructs and myc-CHIP plasmids. 48 h post-transfection, exogenous GST-Dbl were immunoprecipitated from cell lysates by mouse anti-GST Ab, and co-immunoprecipitated CHIP was evaluated by rabbit anti-Myc Ab. The lysate of the NC Cos-7 stable cell line was included as the control (NC). c The expression level of endogenous CHIP, proto-, and onco-Dbl among SKOV-3, GRP75 KD (G2), and NC SKOV-3 stable cell lines were compared by Western blot. Representative blot results from three independent experiments are shown

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques: Stable Transfection, Transfection, Construct, Immunoprecipitation, Control, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: Current results of GRP75 involved in the regulation of proto-Dbl activities are summarized together with previous published data , . See Discussion for details

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques:

Journal: Cell Death & Disease

Article Title: GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway

doi: 10.1038/s41419-018-1039-2

Figure Lengend Snippet: a GRP75 KD (G3) Cos-7 cells were transfected with either GST-proto-Dbl or GST-onco-Dbl plasmids and cultured for 32 h. The cells were then serum-starved for 16 h, lysed, and incubated with GST-fused CRIB (Cdc42 and Rac1 activation) or GST-fused TRBD (RhoA activation) immobilized sepharose beads. The activated Rho GTPases in pull-down precipitates were immunoblotted with corresponding Abs. b COS-7 cells were transfected with GST-Dbl plasmids, serum-starved as described above, and then treated with MKT077 (40 µM, 6 h) before harvest. The activation level changes of Rho GTPases were checked as described above. c GRP75 KD (G3) SKOV-3 cells were transfected with GRP75-EGFP plasmids. SKOV-3 (wt, ctrl), NC SKOV-3, GRP75 KD (G3), and GRP75-EGFP rescued GRP75 KD (G3) cells were serum-starved, lysed, and pull-down for activated Rho GTPase checking as described above. d SKOV-3 (wt, ctrl) cells were treated either with DMSO or with MKT077 (40 µM, 6 h), serum-starved, lysed, and pulled-down for activated Rho GTPase checking as described above. Representative blot results from three independent experiments are shown in a , b , c , d . Protein brands were quantified by Image J software. The activation variation of Rho GTPase proteins were quantified and shown in e , f , g , h , respectively. Statistically significant differences compared with corresponding ctrls ( e ), proto-Dbl transfected NC Cos-7 cells; f , DMSO treated Cos-7 cells; g , h , untransfected SKOV-3 cells (WT or Ctrl)) are shown: ** P < 0.01, * P < 0.05

Article Snippet: Mouse anti-GRP75 Ab (sc-133137), anti-Hsc70 Ab (sc-7298), anti-Hsp90 Ab (sc-13119), anti-Dbl Ab (sc-89), anti-GST Ab (sc-138), anti-CHIP Ab (sc-133066), anti-Cdc42 Ab (sc-8401), anti-RhoA Ab (sc-418), and anti-Rac1 Ab (sc-514583) were obtained from Santa Cruz.

Techniques: Transfection, Cell Culture, Incubation, Activation Assay, Software