Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00246-y

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 for the indicated periods; accompanied by their quantification along with that of α-tubulin ( C ). Bars, 5 μm. * P = 0.02561, *** P = 0.00292; one-sided unpaired Welch’s t test at the indicated time points; n = 6 cells (Ca V 1.2), 17–24 cells (Ca V 2.3), 5–7 cells (α-tubulin). Data are represented by the mean ± SEM. ( D , 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 = 0.0207, one-sided unpaired Welch’s t test, n = 11 cells. Arrows, the timing of plasma membrane fusion. Data are represented by the mean ± SEM. Corresponding to Movie . ( 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 , J ) Proximity ligation assay in CT and cKO primary beta cells with z- projection views, indicating protein binding signals between Ca V 1.2 and the indicated Hsp proteins ( I ); accompanied by quantification ( J ). Scale bar, 5 μm. ** P = 0.0122; *** P = 8.42 × 10 −5 ; one-sided unpaired Welch’s t test, n = 6 cells. Data are represented by the mean ± SEM. ( 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), a mouse anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and a mouse 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, ).

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

Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00246-y

Figure Lengend Snippet: ( A , 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. Scale bar, 5 μm. ns, P = 0.4331; * P = 0.0112; one-way ANOVA with Dunnett’s multiple comparison test, n = 12 cells. Data are represented by the mean ± SEM. ( C , 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 ). ns 1) , P = 0.0634; ns 2) , P = 0.2434; * P = 0.0235; ** 1) P = 0.0014; ** 2) P = 0.0016; ** 1) P = 0.0092; *** P = 3.54 × 10 −4 ; one-sided unpaired Welch’s t test between KD and SC, n = 3–4 biological replicates. Data are represented by the mean ± SEM. 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 , 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 1) , P = 0.4266; ns 2) , P > 0.9999; ** P = 0.0017; one-way ANOVA with Dunnett’s multiple comparison between KD and SC, n = 5–13 biological replicates. Data are represented by the mean ± SEM. 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), a mouse anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and a mouse 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, ).

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

Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00246-y

Figure Lengend Snippet: Reagents and tools table

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), a mouse anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and a mouse 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, ).

Techniques: Knock-Out, Recombinant, Plasmid Preparation, Knockdown, Expressing, Transduction, Sequencing, Software, Enzyme-linked Immunosorbent Assay, Blocking Assay, Transfection, Isolation, Western Blot

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) After 24-h culture of the RCC tumor cells, secreted STIP1 in the culture medium supernatant was detected, while the intracellular protein GAPDH was not detected in the culture medium supernatant indicating no leakage of intracellular components into the culture media. ( B – C ). STIP1 was detected in the purified cell surface protein, while no trace of HSP90 was identified, while HSP90 was detected in the total cell lysates (C). ( D ) Quantification of STIP1 protein in the culture medium supernatant as secreted STIP1 (left panel), and in the purified cell surface protein as outer cell surface STIP1 (right panel). Experiments were triplicated, and mean ± SD was presented. *p < 0.05, vs OS-RC-2; # p < 0.05, vs ACHN. In all panels, western blot images have been cropped to show the protein of interest, and all blots were performed under the same experimental conditions.

Article Snippet: The formalin-fixed, paraffin-embedded tissue sections were stained with a primary mouse anti-human STIP1 monoclonal antibody (1:200, Boster Biotechnology, Wuhan, China) with the Ventana Basic DAB (3,3-diaminobenzidine) Detection kit (Boster Biotechnology, Wuhan, China).

Techniques: Purification, Western Blot

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) STIP1 protein was examined in 19 protein specimens from primary RCC tumors (P, n = 7) and bone metastatic samples (M, n = 12). Proteins were electrophoresed in two 10% SDS-PAGE gels, and were subsequently transferred to two PVDF membranes (10 and 9 specimens for gels 1and 2, respectively). ( B ) The intensity of STIP1 in each lane was normalized with the intensity of GAPDH. *p < 0.05. Experiments were duplicated, and western blot images shown have been cropped to show the protein of interest, and all blots were performed under the same experimental conditions. ( C ) Representative immunohistochemistry staining of STIP1 in primary RCC and bone metastasis tumors. Both intracellular and extracellular STIP1 immunoreactivity was examined as shown in the inset. Images were taken under 20× objective. Scale bar: 50 μm. ( D ) Correlation between the H scores of STIP1 in primary RCC and bone metastasis tumors of the 10 pairs of matched samples. R 2 = 0.6923.

Article Snippet: The formalin-fixed, paraffin-embedded tissue sections were stained with a primary mouse anti-human STIP1 monoclonal antibody (1:200, Boster Biotechnology, Wuhan, China) with the Ventana Basic DAB (3,3-diaminobenzidine) Detection kit (Boster Biotechnology, Wuhan, China).

Techniques: SDS Page, Western Blot, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) STIP1 mRNA expressed highly in the advanced stage RCC tumors. ( B ) STIP1 mRNA expressed highly in the high grades RCC tumors. ( C ) STIP1 mRNA expressed highly in the metastatic RCC tumors (M1+). Overexpression gene rank and P value were generated by the Oncomine algorithms. Fold change was log 2 based.

Article Snippet: The formalin-fixed, paraffin-embedded tissue sections were stained with a primary mouse anti-human STIP1 monoclonal antibody (1:200, Boster Biotechnology, Wuhan, China) with the Ventana Basic DAB (3,3-diaminobenzidine) Detection kit (Boster Biotechnology, Wuhan, China).

Techniques: Over Expression, Generated

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Proliferation of OS-RC-BM5 cells under indicated treatment. ( B ) Cell cycle analysis of OS-RC-BM5 cells under indicated treatment. ( C ) shRNA knockdown of STIP1 in OS-RC-BM5 cells. ( D ) Representative images of Ki67 immunoreactivity in the bone metastasis tumors. Images were taken under 20× objective. Scale bar: 50 μm. ( E ) Correlation between the H scores of STIP1 and percentage of Ki67-positive cells in the same bone metastasis tumors ( n =). R 2 = 0.5868.

Article Snippet: The formalin-fixed, paraffin-embedded tissue sections were stained with a primary mouse anti-human STIP1 monoclonal antibody (1:200, Boster Biotechnology, Wuhan, China) with the Ventana Basic DAB (3,3-diaminobenzidine) Detection kit (Boster Biotechnology, Wuhan, China).

Techniques: Cell Cycle Assay, shRNA, Knockdown

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Representative images of the Transwell membranes with tumor cells migrated to the counter side of the chamber. Note, cell migration was not affected when hrSTIP1 was added into the lower chamber (+ lower chamber). ( B ) Quantification of the migration analysis with three repeats. *p < 0.05, vs vehicle; # p < 0.05, vs hrSTIP1+anti-STIP1.

Article Snippet: The formalin-fixed, paraffin-embedded tissue sections were stained with a primary mouse anti-human STIP1 monoclonal antibody (1:200, Boster Biotechnology, Wuhan, China) with the Ventana Basic DAB (3,3-diaminobenzidine) Detection kit (Boster Biotechnology, Wuhan, China).

Techniques: Migration

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) After 24-h culture of the RCC tumor cells, secreted STIP1 in the culture medium supernatant was detected, while the intracellular protein GAPDH was not detected in the culture medium supernatant indicating no leakage of intracellular components into the culture media. ( B – C ). STIP1 was detected in the purified cell surface protein, while no trace of HSP90 was identified, while HSP90 was detected in the total cell lysates (C). ( D ) Quantification of STIP1 protein in the culture medium supernatant as secreted STIP1 (left panel), and in the purified cell surface protein as outer cell surface STIP1 (right panel). Experiments were triplicated, and mean ± SD was presented. *p < 0.05, vs OS-RC-2; # p < 0.05, vs ACHN. In all panels, western blot images have been cropped to show the protein of interest, and all blots were performed under the same experimental conditions.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Purification, Western Blot

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) STIP1 protein was examined in 19 protein specimens from primary RCC tumors (P, n = 7) and bone metastatic samples (M, n = 12). Proteins were electrophoresed in two 10% SDS-PAGE gels, and were subsequently transferred to two PVDF membranes (10 and 9 specimens for gels 1and 2, respectively). ( B ) The intensity of STIP1 in each lane was normalized with the intensity of GAPDH. *p < 0.05. Experiments were duplicated, and western blot images shown have been cropped to show the protein of interest, and all blots were performed under the same experimental conditions. ( C ) Representative immunohistochemistry staining of STIP1 in primary RCC and bone metastasis tumors. Both intracellular and extracellular STIP1 immunoreactivity was examined as shown in the inset. Images were taken under 20× objective. Scale bar: 50 μm. ( D ) Correlation between the H scores of STIP1 in primary RCC and bone metastasis tumors of the 10 pairs of matched samples. R 2 = 0.6923.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: SDS Page, Western Blot, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) STIP1 mRNA expressed highly in the advanced stage RCC tumors. ( B ) STIP1 mRNA expressed highly in the high grades RCC tumors. ( C ) STIP1 mRNA expressed highly in the metastatic RCC tumors (M1+). Overexpression gene rank and P value were generated by the Oncomine algorithms. Fold change was log 2 based.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Over Expression, Generated

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Proliferation of OS-RC-BM5 cells under indicated treatment. ( B ) Cell cycle analysis of OS-RC-BM5 cells under indicated treatment. ( C ) shRNA knockdown of STIP1 in OS-RC-BM5 cells. ( D ) Representative images of Ki67 immunoreactivity in the bone metastasis tumors. Images were taken under 20× objective. Scale bar: 50 μm. ( E ) Correlation between the H scores of STIP1 and percentage of Ki67-positive cells in the same bone metastasis tumors ( n =). R 2 = 0.5868.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Cell Cycle Assay, shRNA, Knockdown

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Representative images of the Transwell membranes with tumor cells migrated to the counter side of the chamber. Note, cell migration was not affected when hrSTIP1 was added into the lower chamber (+ lower chamber). ( B ) Quantification of the migration analysis with three repeats. *p < 0.05, vs vehicle; # p < 0.05, vs hrSTIP1+anti-STIP1.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Migration

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) ALK2 expression was examined in the cell surface protein of indicated cell lines. ( B ) The Kaplan-Meier curve for overall survival of the TCGA RCC cohort ( n = 88) on the basis of ALK2 mRNA level. Patient was determined as ALK2-high or –low group when the ALK2 expression value was above or below the mean value in the dataset. The survival distributions were estimated by the Kaplan-Meier method, and the significance of differences between survival rates was ascertained using the log-rank test. ( C ) Expressions of p-SMAD1/5, SMAD1/5, ALK2 in the total cell lysate from indicated cell lines upon hrSTIP1 and/or LDN193189, or corresponding vehicle treatment. ( D ) Quantification of the western blot analysis (C) with three repeats. Expression of p-SMAD1/5 was normalized to the level of total SMAD1/5, and ALK2 expression was normalized to the level of GAPDH. *p < 0.05, vs vehicle; # p < 0.05, vs hrSTIP1. ( E ) Knockdown of endogenous SMAD1 and SMAD5 by siRNA decreased the stimulation of pSMAD1/5 protein levels by treatment with 500 nM hrSTIP1. F-G. Suppression of endogenous SMAD1/5 by siRNAs decreased the 96-h proliferation ( F ) and 24-h migration ( G ) of OS-RC-2-BM5 cells under treatment with 500 nM hrSTIP1. Results shown are the mean ± SE from three independent experiments. *p < 0.05, vs control siRNAs + hrSTIP1.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Expressing, Western Blot, Knockdown, Migration, Control

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) TRAP staining of osteoclasts induced in RAW264.7 cell line and primary bone marrow cells upon treatment with hrSTIP1 (500 nM). ( B ) Expressions of CTSK in the total cell lysate from indicated treatment in both RAW264.7 cell line and primary bone marrow cells. ( C ) Quantification of the western blot analysis (B) with three repeats. Expression of CTSK was normalized to the level of β-actin. *p < 0.05, vs vehicle; # p < 0.05, vs hrSTIP1. Western blots images shown are cropped to show the protein of interest, and all blots were performed under the same experimental conditions.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Staining, Western Blot, Expressing

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Expression of PrPc and ALK2 in the total cell lysate from hrSTIP1 treated-RAW264.7 cells. ( B ) TRAP staining of osteoclasts induced in RAW264.7 cell line upon treatment with hrSTIP1 (500 nM) or hrSTIP1+anti-PrPc (10 μg/ml) for 48 hours. ( C ) Expression of CTSK in the total cell lysate from indicated treatment in RAW264.7 cells. ( D ) Expression of p-ERK1/2 and total ERK1/2 in the total cell lysate from indicated treatment in RAW264.7 cells. ( E ) Quantification of the western blot analysis (A, C, D) with three repeats. Expression of PrPc and CTSK were normalized to the level of β-actin, and expression of pERK1/2 was normalized to total ERK1/2. *p < 0.05, vs control; * *p < 0.01, vs RANKL+hrATIP1. ( F ) Suppression the activation of endogenous ERK1/2 signaling by pre-treatment with 15 μM PD98059 for 2-h inhibited the hrSTIP1-induced CTSK protein expression in RAW264.7 cells. ( G ) Quantification of the western blot analysis of CTSK expression with three repeats. *p < 0.05, vs PD98059 -/hrSTIP1+.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Expressing, Staining, Western Blot, Control, Activation Assay

Journal: Disease Models & Mechanisms

Article Title: Hyperactivity and attention deficits in mice with decreased levels of stress-inducible phosphoprotein 1 (STIP1)

doi: 10.1242/dmm.022525

Figure Lengend Snippet: Analyses of mRNA for STIP1 partners in STI1 +/+ , STI1 −/+ and STI1 TGA mouse brains. (A) STIP1 mRNA expression ( n =9 STI1 +/+ , n =5 STI1 TGA and n =4 STI1 −/+ ). (B) PrP C mRNA expression ( n =8 STI1 +/+ , n =4 STI1 TGA and n =7 STI1 −/+ ). (C) Hsp70 mRNA expression ( n =8 STI1 +/+ , n =4 STI1 TGA and n =7 STI1 −/+ ). (D) Hsp90 mRNA expression ( n =3 STI1 +/+ , n =4 STI1 TGA and n =4 STI1 −/+ ). Results are presented as means±s.e.m.; data were analyzed and compared by one-way ANOVA and Bonferroni multiple comparisons post-hoc test; ** P <0.001 and *** P <0.0001 compared with control.

Article Snippet: The antibodies used were anti-STIP1 (1:5000, in-house antibody generated by Bethyl Laboratories Montgomery, USA using recombinant STIP1) , anti-Hsp90 (1:1000), anti-Hsp70 (1:1000), anti-Hsp90α (1:1000), anti Hsp90β (1:1000) (Cell Signaling, Danvers, USA) and anti-PrP 8H4 (1:2000) (Abcam, Cambrige, UK).

Techniques: Expressing, Control

Journal: Disease Models & Mechanisms

Article Title: Hyperactivity and attention deficits in mice with decreased levels of stress-inducible phosphoprotein 1 (STIP1)

doi: 10.1242/dmm.022525

Figure Lengend Snippet: Analyses of protein levels for STIP1 partners in STI1 +/+ , STI −/+ and STI1 TGA mouse brains. (A,B) STIP1 expression in STI1 TGA ( n =9 STI1 +/+ and n =8 STI1 TGA ) and STI1 −/+ mice ( n =8 STI1 +/+ and n =8 STI1 −/+ ). (C,D) PrP C expression in STI1 TGA ( n =6 STI1 +/+ and n =6 STI1 TGA ) and STI1 −/+ mice ( n =6 STI1 +/+ and n =9 STI1 −/+ ). (E,F) Hsp70 expression in STI1 TGA ( n =5 STI1 +/+ and n =4 STI1 TGA ) and STI1 −/+ mice ( n =5 STI1 +/+ and n =4 STI1 −/+ ). (G,H) HSP90 expression in STI1 TGA ( n =10 STI1 +/+ and n =14 STI1 TGA ) and STI1 −/+ mice ( n =6 STI1 +/+ and n =6 STI1 −/+ ). (I,J) Hsp90β ( n =10 STI1 +/+ and n =8 STI1 TGA ) and Hsp90α ( n =5 STI1 +/+ and n =4 STI1 TGA ) in STI1 TGA mice. Results are presented as means±s.e.m.; data were analyzed and compared by Student's t- test; * P <0.05 and *** P <0.0001 compared with control.

Article Snippet: The antibodies used were anti-STIP1 (1:5000, in-house antibody generated by Bethyl Laboratories Montgomery, USA using recombinant STIP1) , anti-Hsp90 (1:1000), anti-Hsp70 (1:1000), anti-Hsp90α (1:1000), anti Hsp90β (1:1000) (Cell Signaling, Danvers, USA) and anti-PrP 8H4 (1:2000) (Abcam, Cambrige, UK).

Techniques: Expressing, Control