Journal: bioRxiv

Article Title: Kinesin-1 mediates proper ER folding of the Ca V 1.2 channel and maintains mouse glucose homeostasis

doi: 10.1101/2024.06.24.600327

Figure Lengend Snippet: (A–C) Degradation assay in immunofluorescence against Ca V 1.2 (A) and Ca V 2.3 (B) of primary beta cells of the indicated genotypes after the CHX treatment of the indicated periods; accompanied by their quantification along with that of α-tubulin (C). Bars, 5 μm. * p < 0.05, *** p < 0.001, Welch’s t test at the indicated time; n = 6 (Ca V 1.2), 17–24 (Ca V 2.3), 5–6 (α-tubulin). (D and E) Brefeldin-A (BFA) washout assay with an LSM 5LIVE-Duo microscope, assessing the speeds of post-Golgi trafficking of Ca V 1.2-EGFP proteins expressed in primary beta cells of the indicated genotypes (D), accompanied by its quantification (E). Time after BFA washout is indicated. Bar, 5 μm. *p < 0.05, Welch’s t test, n = 11. Arrows, the timing of plasma membrane fusion. Corresponding to Movie EV4. (F) TIRF/STORM microscopy of a wild-type primary mouse beta cell immunolabeled against Ca V 1.2 and KIF5B. Scale bar, 5 μm. Arrows, colocalizing spots. (G) TIRF/STORM microscopy of primary mouse beta cells of the indicated genotypes immunolabeled against Ca V 1.2 (green) and Ca V 2.3 (magenta). Scale bars, 5 μm. (H) Schematic representation of STIP1-dependent Hsp70-to-Hsp90 chaperone exchange machinery. (I and J) z -projection of proximity ligation assay in CT and cKO primary beta cells showing the protein binding between Ca V 1.2 and the indicated Hsp proteins (I); accompanied by quantification (J). ** p < 0.01; *** p < 0.001; Welch’s t test, n = 6. (K) Immunoblotting of scramble control (SC) and STIP1-knockdown (KD) MIN6 cells against the indicated epitopes. Note that STIP1 deficiency induced downregulation of Ca V 1.2 and BK Ca proteins. Reproduced twice.

Article Snippet: A rabbit anti-Ca V 1.2 antibody (N-17-R, #sc-16229-R, RRID:AB_2228387), a rabbit anti-K ir 6.2 antibody (H-55; #sc-20809; RRID:AB_2130466), a goat anti-PIP5Kα (PIPKIα) antibody (M-20, #sc-11775; RRID:AB_2268303), and a goat anti-SUR1 antibody (N-18, #sc-11226; RRID:AB_2130475) were purchased from Santa Cruz Biotechnology; a mouse anti-PIP 2 IgM antibody (#Z-A045, RRID:AB_427211) was from Echelon Research labs; a rabbit anti-GFP antibody (#598, RRID:AB_591819) was from MBL; a mouse anti-LC3 antibody (Clone LC3-1703, #CTB-LC3-2-IC, RRID:AB_10707197) was from Cosmo Bio; a rabbit anti-BK Ca (K Ca 1.1) antibody (#APC-151, RRID:AB_10915895) and a rabbit anti-Ca V 2.3 antibody (#ACC-006, RRID:AB_2039777) were from Alomone Labs; a mouse anti-syntaxin-1 antibody (#MAB336, RRID:AB_2196527) was from Millipore; a mouse anti-Na/K ATPase beta 2 antibody (#610914; RRID:AB_398231) and a mouse anti-paxillin antibody (#610051, RRID:AB_397463) were from BD Transduction Labs; a mouse anti-Hsc70/Hsp70 antibody (Clone BB70, #ADI-SPA-822-D, RRID:AB_2039252) and a rat anti-Hsp90 antibody (Clone 16F1, #ADI-SPA-835-D, RRID:AB_2039281) were from Enzo; a rabbit anti-STIP1 antibody (#15218-1-AP; RRID:AB_2255518) and a rabbit anti-calnexin-1 (CANX) antibody (#10427-2-AP, RRID:AB_2069033) were from Proteintech; a mouse anti-derlin-1 antibody (#SAB4200148, RRID:AB_10624068), an anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and an anti-insulin antibody (Clone K36aC10, #I-2018, Sigma Aldrich; RRID:AB_260137) were from Sigma Aldrich; a rabbit anti-phospho Src antibody for pSFK (#ab32078; RRID:AB_2286707) was from Epitomics/Abcam; and a rabbit anti-KIF5B antibody (RRID:AB_2571745) was previously described ( Tanaka et al ., 1998 ).

Techniques: Degradation Assay, Immunofluorescence, Microscopy, Clinical Proteomics, Membrane, Immunolabeling, Proximity Ligation Assay, Protein Binding, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Kinesin-1 mediates proper ER folding of the Ca V 1.2 channel and maintains mouse glucose homeostasis

doi: 10.1101/2024.06.24.600327

Figure Lengend Snippet: (A and B) Rescue of Ca V 1.2 degradation in cKO primary beta cells in the presence of CHX by leupeptin (Leu) or MG-132 (MG) for 4 h. ns, p > 0.05; * p < 0.05; one-way ANOVA, n = 12. (C and D) Vesicle IP of MG-132-treated MIN6 cell lysates transduced with scrambled control (SC) and KIF5B-knockdown (KD) miRNAs, precipitated using Ca V 1.2 or K ir 6.2 antibodies or normal rabbit IgG (NRG) and immunoblotted for the indicated proteins (C), accompanied by quantification of Ca V 1.2-coprecipitated fractions (D). Note that the Ca V 1.2-binding capacities of derlin-1, calnexin-1, and Hsp90 chaperones and that of the adaptor protein STIP1 in KD cell lysates were significantly lower than those in SC cell lysates. (E) Vesicle IP of the MG-132-treated MIN6 cell lysates among the KIF5B KD system against STIP1. Note that the Hsp90 level in the STIP1 immunoprecipitants (IP) was greatly decreased by KIF5B deficiency. Repeated twice. (F) Schematic representation of the working hypothesis on differential KIF5B- and heat-shock-protein (Hsp)-dependencies of opposing ER clients Ca V 1.2 and K ir 6.2 in control (CT) and KIF5B conditional knockout (cKO) mouse beta cells. In cKO cells, Ca V 1.2 fails in chaperone exchange to undergo ERAD-mediated degradation, but K ir 6.2 is intact because it is independent on the KIF5B–Hsp machinery. (G and H) Ca V 1.2 immunocytochemistry of MIN6 cells that had been transduced with EYFP-KIF5B and/or TagRFP-Hsc70 or without them (NT; G); accompanied by their quantification (H). Scale bar, 5 μm. ns, p > 0.05; ** p < 0.01, one-way ANOVA, n = 5–13. Arrow in G, enhanced Ca V 1.2 expression according to dual overexpression. (I) Vesicle IP of non-transduced (NT) and KIF5B- and Hsc70-overexpressing (K5+H70 OE) MIN6 cell lysates against Ca V 1.2. Asterisks, tagged protein bands. The tagRFP-Hsc70 band was overlapped with a band of possibly ubiquitinated form. Reproduced twice.

Article Snippet: A rabbit anti-Ca V 1.2 antibody (N-17-R, #sc-16229-R, RRID:AB_2228387), a rabbit anti-K ir 6.2 antibody (H-55; #sc-20809; RRID:AB_2130466), a goat anti-PIP5Kα (PIPKIα) antibody (M-20, #sc-11775; RRID:AB_2268303), and a goat anti-SUR1 antibody (N-18, #sc-11226; RRID:AB_2130475) were purchased from Santa Cruz Biotechnology; a mouse anti-PIP 2 IgM antibody (#Z-A045, RRID:AB_427211) was from Echelon Research labs; a rabbit anti-GFP antibody (#598, RRID:AB_591819) was from MBL; a mouse anti-LC3 antibody (Clone LC3-1703, #CTB-LC3-2-IC, RRID:AB_10707197) was from Cosmo Bio; a rabbit anti-BK Ca (K Ca 1.1) antibody (#APC-151, RRID:AB_10915895) and a rabbit anti-Ca V 2.3 antibody (#ACC-006, RRID:AB_2039777) were from Alomone Labs; a mouse anti-syntaxin-1 antibody (#MAB336, RRID:AB_2196527) was from Millipore; a mouse anti-Na/K ATPase beta 2 antibody (#610914; RRID:AB_398231) and a mouse anti-paxillin antibody (#610051, RRID:AB_397463) were from BD Transduction Labs; a mouse anti-Hsc70/Hsp70 antibody (Clone BB70, #ADI-SPA-822-D, RRID:AB_2039252) and a rat anti-Hsp90 antibody (Clone 16F1, #ADI-SPA-835-D, RRID:AB_2039281) were from Enzo; a rabbit anti-STIP1 antibody (#15218-1-AP; RRID:AB_2255518) and a rabbit anti-calnexin-1 (CANX) antibody (#10427-2-AP, RRID:AB_2069033) were from Proteintech; a mouse anti-derlin-1 antibody (#SAB4200148, RRID:AB_10624068), an anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and an anti-insulin antibody (Clone K36aC10, #I-2018, Sigma Aldrich; RRID:AB_260137) were from Sigma Aldrich; a rabbit anti-phospho Src antibody for pSFK (#ab32078; RRID:AB_2286707) was from Epitomics/Abcam; and a rabbit anti-KIF5B antibody (RRID:AB_2571745) was previously described ( Tanaka et al ., 1998 ).

Techniques: Transduction, Control, Knockdown, Binding Assay, Knock-Out, Immunocytochemistry, Expressing, Over Expression

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

doi: 10.1016/j.omtm.2018.12.005

Figure Lengend Snippet: The HEPES Method Was Successful for the Transfection of Proteins of Various Molecular Weights, Including Antibodies, Recombinant Proteins, and Small Peptides (A) Anti-STIP1 antibodies were mixed with Opti-MEM (control [Ctrl]) or a pure 20-mM HEPES solution for 15 min before being applied to ARK2 cells cultured in Opti-MEM. After 24 h of incubation, the cells were stained with Alexa Fluor 488-conjugated antibodies and analyzed using a confocal fluorescent microscope. (B) Purified rhSTIP1 proteins were mixed with Opti-MEM (Ctrl) or a pure 20-mM HEPES solution for 15 min before being applied to MDAH2774 cells cultured in Opti-MEM. After 24 h of incubation, the efficiency of rhSTIP1 protein transfection was determined using immunofluorescent staining with anti-v5 antibodies. (C) FITC-Rev peptides were delivered into MOSEC cells using Opti-MEM (Ctrl) or HEPES (30 mM), as described in . After 24 h of incubation, the efficiency of FITC-Rev peptide transfection was determined using immunofluorescent staining. The transfection efficiency was also tested by flow cytometry as these results shown in (D) anti-STIP1 antibodies, (E) rhSTIP1 proteins, or (F) FLAG-Rev peptides. These proteins were preincubated with a pure 20-mM HEPES solution for 15 min before being added to MDAH2774 cells cultured in Opti-MEM. After 24 h of incubation, the delivery efficiency was quantified using flow cytometry. (G) Only the preincubation of protein or peptides with the pure HEPES solution promoted protein transfection. Alexa Fluor 488 anti-mouse IgG antibodies were mixed with only Opti-MEM, HEPES (20 mM), or Tris-HCl (20 mM) for 15 min. Antibody and HEPES mixtures were then added to MDAH2774 cells and cultured in Opti-MEM for another 24 h. To determine whether the sole addition of HEPES into Opti-MEM could promote protein transfection, proteins that had not been preincubated with 20 mM HEPES were added to cells that had been cultured in Opti-MEM with an additional 1.3 mM HEPES, which produced the same final HEPES concentration in the culture medium as in the HEPES-incubating experiments. The transfection efficiency was analyzed using a confocal microscope. (H) Opti-MEM is the most suitable culture medium for the HEPES method. Alexa Fluor 488-conjugated anti-mouse IgG antibodies were delivered to MDAH2774 cells using 20 mM HEPES and cultured for another 24 h. After transfection, the cells were incubated in Opti-MEM (containing 20 mM HEPES), α-MEM (either without HEPES or with an additional 20 mM HEPES), DMEM-F12 (containing 15 mM HEPES), or RPMI 1640 (containing 25 mM HEPES) in the presence and absence of 10% FBS; cells were subsequently analyzed using a confocal fluorescent microscope.

Article Snippet: The antibodies used in this study were Alexa Fluor 546 anti-mouse IgG, Alexa Fluor 488 anti-mouse IgG, v5 (Life Technologies), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), FLAG (Sigma-Aldrich, St. Louis, MO, USA), STIP1 mouse (Abnova, Taipei, Taiwan), and STIP1 rabbit (Gentex, Taipei, Taiwan).

Techniques: Transfection, Recombinant, Control, Cell Culture, Incubation, Staining, Microscopy, Purification, Flow Cytometry, Produced, Concentration Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

doi: 10.1016/j.omtm.2018.12.005

Figure Lengend Snippet: HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p < 0.05. (D) To colocalize transfected proteins with the early endosome, we first used the HEPES method to transfect Alexa Fluor 546-conjugated antibodies into 786-O cells, and then we infected cells with CellLight Early Endosomes-GFP, BacMam 2.0 to express GFP-Rab5. To colocalize transfected proteins with the late endosome, we instead used Alexa Fluor 488-conjugated proteins and CellLight Late Endosomes-RFP, BacMam 2.0 to express RFP-Rab7. After 24 h of incubation, the live 786-O cells were examined using fluorescent microscopy. (E) Self-diffusion coefficients (Ds) were calculated at different molar ratios of STIP1 to HEPES (black squares). The Ds of the NMR internal standard (DSS) obtained under the same experimental conditions served as a reference (red circles). Ctrl, control; Ab, antibody; rh, recombinant human; DSS, dimethyl-silapentane-sulfonic acid.

Article Snippet: The antibodies used in this study were Alexa Fluor 546 anti-mouse IgG, Alexa Fluor 488 anti-mouse IgG, v5 (Life Technologies), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), FLAG (Sigma-Aldrich, St. Louis, MO, USA), STIP1 mouse (Abnova, Taipei, Taiwan), and STIP1 rabbit (Gentex, Taipei, Taiwan).

Techniques: Transfection, Neutralization, Incubation, Microscopy, Software, Lactate Dehydrogenase Assay, Infection, Diffusion-based Assay, Control, Recombinant

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

doi: 10.1016/j.omtm.2018.12.005

Figure Lengend Snippet: The Efficient HEPES-Mediated Transfection of Antibodies into the Cytosol Mouse IgG 2 antibodies (Ctrl IgG) or anti-STIP1 antibodies were preincubated in a pure 30-mM HEPES solution for 15 min before being applied to HeLa cells. After 24 h of incubation, STIP1 protein levels were analyzed using western blotting with an anti-STIP1 rabbit antibody. IgG heavy chains were detected with donkey anti-mouse IgG antibodies. Error bars indicate the SEM (n = 3). **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies used in this study were Alexa Fluor 546 anti-mouse IgG, Alexa Fluor 488 anti-mouse IgG, v5 (Life Technologies), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), FLAG (Sigma-Aldrich, St. Louis, MO, USA), STIP1 mouse (Abnova, Taipei, Taiwan), and STIP1 rabbit (Gentex, Taipei, Taiwan).

Techniques: Transfection, Incubation, Western Blot

Journal: Genes, chromosomes & cancer

Article Title: Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer

doi: 10.1002/gcc.22136

Figure Lengend Snippet: STIP1 is highly expressed in human ovarian cancer cells and tissue specimens. A. STIP1 protein levels were analyzed by western blot. GAPDH was included as an internal loading control. B. Representative immunohistochemical staining for STIP1 in formalin-fixed paraffin-embedded EOC tissues (400×). C. IHC staining score of STIP1 in EOC samples was significantly higher than that in healthy controls, benign ovarian tumors, or borderline ovarian tumors (P < 0.001, P < 0.001, and P = 0.039 respectively). Differences between tumor stages and grades were statistically significant, with advanced-stage and poor-grade specimens having higher immunoreactivity.

Article Snippet: The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Western Blot, Control, Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Genes, chromosomes & cancer

Article Title: Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer

doi: 10.1002/gcc.22136

Figure Lengend Snippet: Expression of STIP1 in Relation to Clinicopathological Characteristics in IHC Analysis

Article Snippet: The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Expressing, Diagnostic Assay

Journal: Genes, chromosomes & cancer

Article Title: Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer

doi: 10.1002/gcc.22136

Figure Lengend Snippet: Kaplan-Meier plots for patients with EOC stratified according to tumor stage, STIP1 expression, tumor grade, or CA125 expression.

Article Snippet: The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Expressing

Journal: Genes, chromosomes & cancer

Article Title: Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer

doi: 10.1002/gcc.22136

Figure Lengend Snippet: Univariate and Multivariate Analyses of the Associations Between Prognostic Variables and Overall Survival in 113 Cases of Epithelial Ovarian Cancer

Article Snippet: The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C.

Techniques:

Journal: Genes, chromosomes & cancer

Article Title: Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer

doi: 10.1002/gcc.22136

Figure Lengend Snippet: STIP1 knockdown by siRNA in EOC cells. Whole cell lysates and total RNA were collected from SKOV3, OVCAR3, or YDOV-13 cells following STIP1 knockdown by siRNA. Expression of STIP1 mRNA was measured by real-time PCR (A) and protein level was analyzed by immunoblotting (B). β-actin mRNA levels were used to normalize STIP1 mRNA expression. An asterisk (*) indicates a P value <0.05.

Article Snippet: The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Genes, chromosomes & cancer

Article Title: Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer

doi: 10.1002/gcc.22136

Figure Lengend Snippet: Effects of STIP1 on cell proliferation, migration, and invasiveness of EOC cells. A. Cell proliferation of SKOV3, OVCAR3, and YDOV-13 was determined by MTT assay 72 h after siRNA transfection. Error bars represent the SD of triplicate experiments. Cell migration (B) and invasion (C) analyses of SKOV3 cells were performed. Migrating or invasive cells were quantified using a plate reader at OD 560 nm. D. STIP1 knockdown (siSTIP1#1 and siSTIP1#2) decreased mRNA level of ID3 in OVCAR3 cells. E. OVCAR3 cells were assayed for phospho-Smad1/Smad5. STIP1 knockdown (siSTIP1#1 and siSTIP1#2) reduced phosphorylation of Smad1/Smad5. The total amount of Smad5 and actin was used as the protein loading control. An asterisk (*) indicates a P value <0.05.

Article Snippet: The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Migration, MTT Assay, Transfection, Knockdown, Phospho-proteomics, Control

Journal: Genes, chromosomes & cancer

Article Title: Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer

doi: 10.1002/gcc.22136

Figure Lengend Snippet: STIP1 knockdown inhibits in vivo xenograft tumor growth. For the xenograft tumor growth assay, treatments were started 1 week after tumor cell injection, and siControl or siSTIP1 was injected every 3 days for 3 weeks at a dose of 5 mg/kg body weight. A. STIP1 knockdown reduces the growth rate of xenografted SKOV3 cells in BALB/c-nu mice. Mean tumor volume ± SD for each group was calculated at each week. B. Ki-67, BCL2, Tp53, and Bax mRNA levels were analyzed by qRT-PCR and normalized to β-actin mRNA levels. An asterisk (*) indicates a P value <0.05.

Article Snippet: The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Knockdown, In Vivo, Growth Assay, Injection, Quantitative RT-PCR