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: Journal of Cellular and Molecular Medicine

Article Title: Stress‐induced phosphoprotein 1 restrains spinal cord ischaemia‐reperfusion injury by modulating NF‐κB signalling

doi: 10.1111/jcmm.17030

Figure Lengend Snippet: Primary antibodies used for Western blotting assay

Article Snippet: The affinity agarose beads were pre‐coated with mouse anti‐human STIP1 monoclonal antibody (lot number: sc‐393475; Santa Cruz Biotechnology) and moue anti‐human HSPA8 (lot number: sc‐7298; Santa Cruz Biotechnology) or IgG antibody.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Stress‐induced phosphoprotein 1 restrains spinal cord ischaemia‐reperfusion injury by modulating NF‐κB signalling

doi: 10.1111/jcmm.17030

Figure Lengend Snippet: STIP1 expression increases firstly and then decreases in rats after SCII treatment. A, The rats undergo ischaemia by clamping abdominal aorta for 1 h and reperfusion for 24 h; B, Western blotting detects STIP1 expression in rat spinal cord specimens in absence or presence of SCII treatment; C, Immunohistochemical staining detects STIP1 expression in rat spinal cord specimens. The brown represents STIP1, and the STIP1‐postive cells are indicated by arrows. Scale bar =50 μm. SCII, spinal cord ischaemia‐reperfusion injury; STIP1, stress‐induced phosphoprotein 1. ** p < 0.01, *** p < 0.001 versus the Sham group

Article Snippet: The affinity agarose beads were pre‐coated with mouse anti‐human STIP1 monoclonal antibody (lot number: sc‐393475; Santa Cruz Biotechnology) and moue anti‐human HSPA8 (lot number: sc‐7298; Santa Cruz Biotechnology) or IgG antibody.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Stress‐induced phosphoprotein 1 restrains spinal cord ischaemia‐reperfusion injury by modulating NF‐κB signalling

doi: 10.1111/jcmm.17030

Figure Lengend Snippet: STIP1 alleviates ischaemia/reperfusion‐induced motor function impairment and neuronal injury in rat spinal cord. A, Ischaemia/reperfusion is given to rats; B, Western blotting detects STIP1 expression in rat spinal cord specimens after ST1P1 overexpression; C, BBB scoring measures the motor function of rat hind limbs; D, HE staining detects the morphological changes of rat spinal cord. Scale bar =100 μm; E, immunohistochemical staining detects the expression of the neuron marker NeuN. Scale bar =50 μm. BBB, Basso, Beattie and Bresnahan; *** p < 0.001 versus the Sham group, ### p < 0.001 versus the SCII + LV group

Article Snippet: The affinity agarose beads were pre‐coated with mouse anti‐human STIP1 monoclonal antibody (lot number: sc‐393475; Santa Cruz Biotechnology) and moue anti‐human HSPA8 (lot number: sc‐7298; Santa Cruz Biotechnology) or IgG antibody.

Techniques: Western Blot, Expressing, Over Expression, Staining, Immunohistochemical staining, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Stress‐induced phosphoprotein 1 restrains spinal cord ischaemia‐reperfusion injury by modulating NF‐κB signalling

doi: 10.1111/jcmm.17030

Figure Lengend Snippet: STIP1 ameliorates ischaemia/reperfusion‐induced inflammation in rat spinal cord. A, Immunohistochemical staining determines the expression and location of Iba‐1 and STIP1. The arrows indicate co‐location of STIP1 and Iba‐1. Scale bar =50 μm; B, ELISA detects TNF‐α and IL‐6 secretion in rat spinal cord specimens following SCII treatment and STIP1 overexpression; C, Western blotting determines IκBβ expression in rat spinal cord specimens; D, Western blotting determines the cytoplasmic and nuclear expression of NF‐κB p65. NF‐κB, nuclear factor kappa B; SCII, spinal cord ischaemia‐reperfusion injury; STIP1, stress‐induced phosphoprotein 1; TNF, tumour necrosis factor. *** p < 0.001 versus the Sham group; # p < 0.05, ### p < 0.001 versus the SCII + LV group

Article Snippet: The affinity agarose beads were pre‐coated with mouse anti‐human STIP1 monoclonal antibody (lot number: sc‐393475; Santa Cruz Biotechnology) and moue anti‐human HSPA8 (lot number: sc‐7298; Santa Cruz Biotechnology) or IgG antibody.

Techniques: Immunohistochemical staining, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Stress‐induced phosphoprotein 1 restrains spinal cord ischaemia‐reperfusion injury by modulating NF‐κB signalling

doi: 10.1111/jcmm.17030

Figure Lengend Snippet: STIP1 restrains OGD‐induced inflammation in microglial cells. A, BV2 cell treatments; B, Western blotting determines STIP1 expression in BV2 cells after OGD treatment, C, Immunoprecipitation detects the binding between STIP1 and HSPA8 in BV2 cells; D, Western blotting determines STIP1 expression after STIP1 overexpression; E, Immunoprecipitation detects the interaction between HSPA8 and IκBβ in BV2 cells; F, Western blotting detects IκBβ expression in BV2 cells; G, Western blotting detects cytoplasmic and nuclear NF‐κB p65 levels in BV2 cells; H, Immunofluorescent staining detects the location of NF‐κB p65. Scale bar =50 μm; I, Immunofluorescent staining detects the expression of the microglia marker Iba‐1. Scale bar =50 μm; J, ELISA detects the secretion of inflammatory factors TNF‐α and IL‐6 in the cell culture supernatant; K, Western blotting determines TNF‐α and IL‐6 expression in BV2 cells after OGD treatment and STIP1 overexpression. NF‐κB, nuclear factor kappa B; OGD, oxygen and glucose deprivation; SCII, spinal cord ischaemia‐reperfusion injury; STIP1, stress‐induced phosphoprotein 1; TNF, tumour necrosis factor. ** p < 0.01, *** p < 0.001 versus the Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 versus the SCII + LV group

Article Snippet: The affinity agarose beads were pre‐coated with mouse anti‐human STIP1 monoclonal antibody (lot number: sc‐393475; Santa Cruz Biotechnology) and moue anti‐human HSPA8 (lot number: sc‐7298; Santa Cruz Biotechnology) or IgG antibody.

Techniques: Western Blot, Expressing, Immunoprecipitation, Binding Assay, Over Expression, Staining, Marker, Enzyme-linked Immunosorbent Assay, Cell Culture

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: Knockdown of STIP1 was achieved by a validated hairpin STIP1 shRNA Lentiviral Particle Gene Silencer kit (Santa Cruz Biotechnology, Texas, USA).

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: Knockdown of STIP1 was achieved by a validated hairpin STIP1 shRNA Lentiviral Particle Gene Silencer kit (Santa Cruz Biotechnology, Texas, USA).

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: Knockdown of STIP1 was achieved by a validated hairpin STIP1 shRNA Lentiviral Particle Gene Silencer kit (Santa Cruz Biotechnology, Texas, USA).

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: Knockdown of STIP1 was achieved by a validated hairpin STIP1 shRNA Lentiviral Particle Gene Silencer kit (Santa Cruz Biotechnology, Texas, USA).

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: Knockdown of STIP1 was achieved by a validated hairpin STIP1 shRNA Lentiviral Particle Gene Silencer kit (Santa Cruz Biotechnology, Texas, USA).

Techniques: Migration

Journal: International Journal of Molecular Sciences

Article Title: TFIP11, CCNL1 and EWSR1 Protein-protein Interactions, and Their Nuclear Localization

doi: 10.3390/ijms9081504

Figure Lengend Snippet: Northern blot analysis. Northern blot analysis of TFIP11, CCNL1 and EWSR1 in multiple organ tissues. Size markers in kb are shown on the left.

Article Snippet: Full-length cDNAs for human TFIP11, CCNL1 and EWSR1 were purchased from Origene (Rockville, MD; catalogue #s SC112654/NM_012143, TC113160/NM_020307 and TC116868/NM_005243 respectively).

Techniques: Northern Blot

Journal: International Journal of Molecular Sciences

Article Title: TFIP11, CCNL1 and EWSR1 Protein-protein Interactions, and Their Nuclear Localization

doi: 10.3390/ijms9081504

Figure Lengend Snippet: Colocalization of TFIP11 and CCNL1, and TFIP11 and EWSR1. HeLa cells transfected with CCNL1-N1 (panel A) or EWSR1-Red-N1 (panel B) and were fixed and labeled with anti-TFIP11 antibody. Endogenous TFIP11 (eTFIP11) localization (using Alexa Fluor 568 goat anti-rabbit antibody; panel Ai) was compared to transfected CCNL1 (tCCNL1) localization (panel Aii) in the merged image (panel Aiii). Endogenous TFIP11 localization (using Alexa Fluor 488 goat anti-rabbit antibody; panel Bi) was compared to transfected EWSR1 (tEWSR1) localization (panel Bii) in the merged image (panel Biii). Cell nuclei are stained blue by DAPI (panels Aiv and Biv). Scale bars, 10 μm.

Article Snippet: Full-length cDNAs for human TFIP11, CCNL1 and EWSR1 were purchased from Origene (Rockville, MD; catalogue #s SC112654/NM_012143, TC113160/NM_020307 and TC116868/NM_005243 respectively).

Techniques: Transfection, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: TFIP11, CCNL1 and EWSR1 Protein-protein Interactions, and Their Nuclear Localization

doi: 10.3390/ijms9081504

Figure Lengend Snippet: EWSR1 interacts with TFIP11. HEK293 cells were transfected with plasmid expressing EWSR1-Myc (lanes 1, 3, 5 and 7), or EWSR1-Myc and TFIP-FLAG (lanes 2, 4, 6 and 8). The input panels (lanes 1, 2, 5 and 6) show protein in the cell lysate prior to immunoprecipitation. Cell lysates were immunoprecipitated using anti-FLAG monoclonal antibody (lanes 3, 4, 7 and 8), and then immunoblotted with either the anti-FLAG (lanes 1–4) or anti-Myc antibody (lanes 5–8). The plasmid pcDNA was cotransfected as a blank control to ensure equal concentrations of plasmid DNA being used for each transfection. Immunoprecipitate (IP), Immunoblot (IB).

Article Snippet: Full-length cDNAs for human TFIP11, CCNL1 and EWSR1 were purchased from Origene (Rockville, MD; catalogue #s SC112654/NM_012143, TC113160/NM_020307 and TC116868/NM_005243 respectively).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot

Journal: BMC Cancer

Article Title: Inhibition of STAT3-interacting protein 1 (STATIP1) promotes STAT3 transcriptional up-regulation and imatinib mesylate resistance in the chronic myeloid leukemia

doi: 10.1186/1471-2407-14-866

Figure Lengend Snippet: Immunofluorescence analyses of STAT3, STAT3-Y and STATIP1 proteins. STAT3, STAT3-Y and STATIP1 FITC-labeled antibodies (green), DAPI-stained DNA (blue) and merged images. Protein labeling was observed in untreated K562 cells (A-I) and K562 cells treated with 1 μM IM (J-R) . The slides were analyzed using an LMS confocal system, and the images were processed using AxioVision-LE software (Carl Zeiss).

Article Snippet: STATIP1 siRNA (100 nM) (SC-44436, Santa Cruz) and 2 μL of LipofectamineTM RNAiMAX (Invitrogen) were incubated separately in a final volume of 50 μL of RPMI-1640 media for 5 min.

Techniques: Immunofluorescence, Labeling, Staining, Software

Journal: BMC Cancer

Article Title: Inhibition of STAT3-interacting protein 1 (STATIP1) promotes STAT3 transcriptional up-regulation and imatinib mesylate resistance in the chronic myeloid leukemia

doi: 10.1186/1471-2407-14-866

Figure Lengend Snippet: Expression levels of STATIP1 , STAT3 , CCND1 and BCL-XL genes in response to IM/LLL-3 treatments. (A) Relative mRNA levels of STATIP1, STAT3, CCND1 and BCL-XL after 24 h of 1 μM IM treatment. (B) Relative mRNA levels of STATIP1, STAT3, CCND1 and BCL-XL after 24 h of 40 μM LLL-3 treatment. (C) Western blot analysis of STAT3 and STATIP1 protein levels and STAT3-Y705 phosphorylation 24 h after 1 μM IM treatment. (D) The protein levels were determined by densitometry analysis in ImageJ software version 1.44. All comparisons were made to untreated cells – ctrl. Ctrl: control. The data represent the mean ± SD of at least three independent experiments (*p <0.05 and **p <0.01).

Article Snippet: STATIP1 siRNA (100 nM) (SC-44436, Santa Cruz) and 2 μL of LipofectamineTM RNAiMAX (Invitrogen) were incubated separately in a final volume of 50 μL of RPMI-1640 media for 5 min.

Techniques: Expressing, Western Blot, Phospho-proteomics, Software, Control

Journal: BMC Cancer

Article Title: Inhibition of STAT3-interacting protein 1 (STATIP1) promotes STAT3 transcriptional up-regulation and imatinib mesylate resistance in the chronic myeloid leukemia

doi: 10.1186/1471-2407-14-866

Figure Lengend Snippet: STATIP1 mRNA depletion by siRNA induces the over-expression of STAT3 and its target genes. (A) STATIP1 mRNA levels at 24 h, 48 h and 72 h after STATIP1 silencing, as determined by RT-qPCR. (B) Western blot analyses of the STATIP1 protein level 72 h after STATIP1 silencing. (C) RT-qPCR analyses of the STAT3 mRNA levels at 24 h, 48 h and 72 h after STATIP1 silencing. (D) RT-qPCR analyses of the CCND1 and BCL-XL mRNA levels at 72 h after STATIP1 silencing. All comparisons were made to untreated cells – ctrl and scrambled-treated cells. Ctrl: control. The data represent the means ± SD of at least three independent experiments (*p <0.05 and **p <0.01).

Article Snippet: STATIP1 siRNA (100 nM) (SC-44436, Santa Cruz) and 2 μL of LipofectamineTM RNAiMAX (Invitrogen) were incubated separately in a final volume of 50 μL of RPMI-1640 media for 5 min.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Control

Journal: BMC Cancer

Article Title: Inhibition of STAT3-interacting protein 1 (STATIP1) promotes STAT3 transcriptional up-regulation and imatinib mesylate resistance in the chronic myeloid leukemia

doi: 10.1186/1471-2407-14-866

Figure Lengend Snippet: Involvement of STATIP1 and STAT3 genes in IM resistance in CML cell lines. (A) STATIP1 mRNA levels in K562 and Lucena cells determined by RT-qPCR. (B) STAT3 mRNA levels, as determined by RT-qPCR, in Lucena cells under the following conditions: 1 μM IM treatment, 40 μM LLL-3 treatment, and co-treatment after 24 h. (C) The ABCB1 mRNA levels in Lucena cells were determined by RT-qPCR under the treatment conditions noted above. (D) Apoptotic cells were measured by flow cytometry in both cell lines under the treatment conditions noted above. The cell cycle was evaluated by flow cytometry after being subjected to the treatment conditions noted above in K562 (E) and Lucena (F) cells. All comparisons were made to untreated cells – ctrl. Ctrl: control. The data represent the mean ± SD of at least three independent experiments (*p <0.05 and **p <0.01).

Article Snippet: STATIP1 siRNA (100 nM) (SC-44436, Santa Cruz) and 2 μL of LipofectamineTM RNAiMAX (Invitrogen) were incubated separately in a final volume of 50 μL of RPMI-1640 media for 5 min.

Techniques: Quantitative RT-PCR, Flow Cytometry, Control

Journal: BMC Cancer

Article Title: Inhibition of STAT3-interacting protein 1 (STATIP1) promotes STAT3 transcriptional up-regulation and imatinib mesylate resistance in the chronic myeloid leukemia

doi: 10.1186/1471-2407-14-866

Figure Lengend Snippet: Evaluation of cell viability in STATIP1-silenced K562 cells after IM treatment. The relative percentage of viable cells was determined by WST-1 assay after 24 h of IM treatment (+), compared to untreated cells – ctrl and scrambled-treated cells. Ctrl: control. The data represent the mean ± SD of at least three independent experiments (*p <0.05 and **p <0.01).

Article Snippet: STATIP1 siRNA (100 nM) (SC-44436, Santa Cruz) and 2 μL of LipofectamineTM RNAiMAX (Invitrogen) were incubated separately in a final volume of 50 μL of RPMI-1640 media for 5 min.

Techniques: WST-1 Assay, Control

Journal: BMC Cancer

Article Title: Inhibition of STAT3-interacting protein 1 (STATIP1) promotes STAT3 transcriptional up-regulation and imatinib mesylate resistance in the chronic myeloid leukemia

doi: 10.1186/1471-2407-14-866

Figure Lengend Snippet: Expression levels of STATIP1 and STAT3 genes in CML patients. (A) STATIP1 mRNA levels and (B) STAT3 mRNA levels were determined by RT-qPCR analyses in 6 IM-responsive patients and 8 IM-resistant patients. Raw expression values were normalized to β-actin expression. Expression changes were calibrated by 6 healthy bone marrow donors analysis. Resp. P = responsive patients; Resist. P. = resistant patients. (*p <0.05).

Article Snippet: STATIP1 siRNA (100 nM) (SC-44436, Santa Cruz) and 2 μL of LipofectamineTM RNAiMAX (Invitrogen) were incubated separately in a final volume of 50 μL of RPMI-1640 media for 5 min.

Techniques: Expressing, Quantitative RT-PCR