Journal: International Journal of Molecular Sciences

Article Title: STIM1 Deficiency Leads to Specific Down-Regulation of ITPR3 in SH-SY5Y Cells

doi: 10.3390/ijms21186598

Figure Lengend Snippet: Downregulation of ITPR3 in STIM1-deficient cells. ( a ) Total RNA was purified from undifferentiated wild-type (WT) and STIM1-KO cells, and the quantification of transcripts was evaluated from a Taqman Gene Expression Array (Ref. #4418932). Data from 3 different experiments are given in the (n = 3 for WT; n = 3 for KO). From these data, the threshold cycle (Ct) was calculated to evaluate the expression fold change as 2^(−ΔΔC t ) which is also plotted in the bar chart, as the fold-change of ITPR1/3 expression in STIM1-KO cells compared with wild-type cells. Expression of GAPDH was used as a housekeeping gene. ( b ) Quantification of ITPR1/2/3 transcripts was performed individually with specific primers (see Methods, ). The bar chart depicts the expression fold change of transcripts in STIM1-KO cells compared with wild-type cells. Data are mean ± S.D. from 2 different biological replicates with technical triplicates (n = 6). ( c – e ) ITPR3, ITPR1, and ITPR2 protein expression in whole cell lysates was quantified in wild-type and STIM1-KO cells. Beta-tubulin was used as a loading control. Data are mean ± S.D. from 3 independent experiments (for ITPR1) or 4 independent experiments (for ITPR2 and ITPR3). ( f ) ITPR3 protein expression (left panel) or total levels of ITPRs (right panel) were evaluated in osteosarcoma U2OS cells, as well as in wild-type and STIM1-KO SH-SY5Y cells by immunoblot. Beta-tubulin was used as a loading control. (***) Statistical significance p < 0.001.

Article Snippet: The rabbit polyclonal anti-STIM1 antibody (#4119) was from ProSci Inc. (Poway, CA, USA); the mouse monoclonal anti-red fluorescent protein (RFP, clone 6G6) was from Chromotek (Planegg-Martinsried, Germany); the mouse anti-beta tubulin antibody (clone TUB2.1) was from Sigma-Aldrich; the mouse anti-ITPR3 antibody (#610312), was from BD Biosciences (Franklin Lakes, NJ, USA); the mouse anti-ITPR2 antibody (sc-398434, clone A-5), the mouse anti-ITPR1/2/3 antibody (sc-377518, clone B-2), the mouse anti-ASCL4 (sc-365230, clone F-4), mouse anti-VDAC1 (sc-390996, clone B-6), and the mouse monoclonal anti-GAPDH antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany); the rabbit anti-ITPR1 antibody (RBT-03) was a kind gift from Dr. Jan B. Parys (KU Leuven) who generated and characterized the antibody elsewhere [ ].

Techniques: Purification, Gene Expression, Expressing, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: STIM1 Deficiency Leads to Specific Down-Regulation of ITPR3 in SH-SY5Y Cells

doi: 10.3390/ijms21186598

Figure Lengend Snippet: Downregulation of ITPR3 in differentiated STIM1-KO cells. ( a ) Quantification of ITPR1 , ITPR2 , and ITPR3 transcripts from differentiated STIM1-KO cells compared to wild-type cells. Data are mean ± S.D. from 2 different biological replicates with technical triplicates (n = 6). ( b ) ITPR3 protein expression in whole cell lysates was quantified in differentiated wild-type and STIM1-KO cells (beta-tubulin as a loading control). Data are mean ± S.D. from 2 independent experiments.

Article Snippet: The rabbit polyclonal anti-STIM1 antibody (#4119) was from ProSci Inc. (Poway, CA, USA); the mouse monoclonal anti-red fluorescent protein (RFP, clone 6G6) was from Chromotek (Planegg-Martinsried, Germany); the mouse anti-beta tubulin antibody (clone TUB2.1) was from Sigma-Aldrich; the mouse anti-ITPR3 antibody (#610312), was from BD Biosciences (Franklin Lakes, NJ, USA); the mouse anti-ITPR2 antibody (sc-398434, clone A-5), the mouse anti-ITPR1/2/3 antibody (sc-377518, clone B-2), the mouse anti-ASCL4 (sc-365230, clone F-4), mouse anti-VDAC1 (sc-390996, clone B-6), and the mouse monoclonal anti-GAPDH antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany); the rabbit anti-ITPR1 antibody (RBT-03) was a kind gift from Dr. Jan B. Parys (KU Leuven) who generated and characterized the antibody elsewhere [ ].

Techniques: Expressing, Control

Journal: Cancers

Article Title: Potential Role of PDGFRβ-Associated THBS4 in Colorectal Cancer Development

doi: 10.3390/cancers12092533

Figure Lengend Snippet: Effect of PDGF-D stimulation of THBS4 after blockage of PDGFRβ, IP3R, and STIM1. ( A ) Western blot with anti-THBS4 antibody in whole cell lysate (left panel) or cultured medium (right panel) of DLD-1 cells cultured with PDGF-D (20 μM) for 8 h in the presence of imatinib (0, 0.2, 0.5, 1, 2, and 5 μM) for 16 h, 2-APB (0, 5, 10, 20, 50, and 100 μM) for 16 h, or ML-9 (0, 5, 10, 20, 50, and 100 μM) for 16 h, respectively. ( B ) Western blot with anti-THBS4 antibody in whole cell lysate or cultured medium of DLD-1 cells transfected with siIP3R or siSTIM1 and cultured with PDGF-D (20 μM) for 8 h. ( C ) Relative mRNA expression levels as determined with real-time PCR for THBS4 of DLD-1 cells cultured with PDGF-D (20 μM) for 8 h in the presence of imatinib (5 μM) for 16 h, 2-APB (100 μM) for 16 h or ML-9 (100 μM) for 16 h, respectively. Three independent experiments were performed in duplicate. * p < 0.05 when compared with the control t- test.

Article Snippet: DLD-1 cells were transfected with IP3R siRNA 10nM (sc-42475, Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA) or STIM1 siRNA 10 nM (sc-76589) along with control siRNA (sc-37007).

Techniques: Western Blot, Cell Culture, Transfection, Expressing, Real-time Polymerase Chain Reaction, Control