negative control scrambled sirna  (Santa Cruz Biotechnology)

Journal: Autophagy Reports

Article Title: TRPML3 and TRPML1/3 differentially regulate unconventional MIF secretion through TMED10 and secretory autophagy

doi: 10.1080/27694127.2026.2740425

Figure Lengend Snippet: Relationship between MIF and TMED10. (A) Secretion of MIF was measured in HEK293T cells either untreated or treated with brefeldin A. (B) Secretion of MIF was measured in HEK293T cells untreated or treated with the indicated doses of probenecid. (C) GST or the indicated GST-TMED10 domains coupled to glutathione sepharose beads were incubated with whole cell lysates from HEK293T cells expressing Flag-MIF. Elutes were subjected to SDS-PAGE and immunoblotted with an anti-Flag antibody. (D) GST-MIF coupled to glutathione sepharose beads was incubated with whole cell lysate from HEK293T cells expressing GFP-TMED10 or GFP-TMED10(ΔCT). Elutes were subjected to SDS-PAGE and immunoblotted with anti-GFP antibody. (E) Secretion of MIF was measured in HEK293T cells expressing mock or GFP-TMED10. (F) Secretion of MIF was measured in HEK293T cells treated with control siRNA or siRNA targeting TMED10 for 72 h. (G) A crosslink assay was performed in HEK293T cells expressing GFP-TMED10 with mock or Flag-MIF to examine the oligomeric form of TMED10. (H) Secretion of Flag-MIF was measured in HEK293T cells expressing mock, RAB1A, 1B, and 2A.

Article Snippet: For knockdown experiments, siRNA targeting human TMED10 (BIONEER, 10,972–1), human ATG5 (BIONEER, 9474–1), human SYT5 (BIONEER, 6861–1), and a negative control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) were used.

Techniques: Incubation, Expressing, SDS Page, Control

Journal: Autophagy Reports

Article Title: TRPML3 and TRPML1/3 differentially regulate unconventional MIF secretion through TMED10 and secretory autophagy

doi: 10.1080/27694127.2026.2740425

Figure Lengend Snippet: Effect of autophagy on MIF secretion. (A) HeLa cells expressing GFP-LC3 and mCherry-MIF were treated with cold digitonin (50 µg/ml) and imaged by fluorescence microscopy. (B) Secretion of MIF was measured in HEK293T cells either untreated or treated with 10 μM SAR405. (C) Secretion of MIF was measured in HEK293T cells treated with control siRNA or siRNA targeting ATG5 for 72 h. (D) Secretion of MIF was measured in HEK293T cells either untreated or treated with 100 nM bafilomycin A1. (E) Secretion of MIF was measured in HEK293T cells treated with control siRNA or siRNA targeting SYT5 for 48 h. Knockdown efficiency was confirmed by immunoblotting for SYT5. (F) Secretion of Flag-MIF was measured in HEK293T cells expressing mock or GFP-SEC22B. (G) HeLa cells expressing GFP-SEC22B and mCherry-MIF were treated with cold digitonin (50 µg/ml) and imaged by fluorescence microscopy. (H, upper) HeLa cells co-expressing GFP-MIF and Flag-VAMP4 were treated with cold digitonin (50 µg/ml), immunostained with an anti-Flag antibody, and imaged by fluorescence microscopy. (H, lower) HeLa cells expressing GFP-VAMP8 and mCherry-MIF were treated with cold digitonin (50 µg/ml) and imaged by fluorescence microscopy.

Article Snippet: For knockdown experiments, siRNA targeting human TMED10 (BIONEER, 10,972–1), human ATG5 (BIONEER, 9474–1), human SYT5 (BIONEER, 6861–1), and a negative control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) were used.

Techniques: Expressing, Fluorescence, Microscopy, Control, Knockdown, Western Blot

Journal: Bioscience Reports

Article Title: G3BP1 is a SWI/SNF-bound regulator of transcription that modulates activation of STATs

doi: 10.1042/BSR20250290

Figure Lengend Snippet: ( A, B ) G3BP1 was transiently silenced with siRNA, or not; cells were then serum-starved and stimulated with PDGF-BB, as indicated. Activation of PDGFR, STAT1, ERK1/2, AKT, PLCγ, and STAT3 signaling pathways was determined by immunoblotting for phosphorylated and total amounts of the signaling proteins. The experiments were repeated four times.

Article Snippet: AG01523 cells were transiently transfected with 20 nM siRNA of Trilencer-27 G3BP siRNA (#SR, OriGene Technologies, U.S.A.) or 20 nM scrambled negative control siRNA (#SR30004, OriGene Technologies, U.S.A.), using SilentFect reagent (Bio-Rad), and incubated for 72 to 96 h at 37°C in a CO 2 incubator.

Techniques: Activation Assay, Protein-Protein interactions, Western Blot

Journal: Bioscience Reports

Article Title: G3BP1 is a SWI/SNF-bound regulator of transcription that modulates activation of STATs

doi: 10.1042/BSR20250290

Figure Lengend Snippet: (A–F) After transient silencing of G3BP1 with siRNA, or not, and serum starvation overnight, AG01523 cells were stimulated with PDGF-BB for 1 h (black bars) or left unstimulated (0 h, gray bars). mRNA expression of G3BP1 ( A ), STAT3 ( B ), STAT1 ( C ), FOS ( D ), MYC ( E ), and CCND1 (cyclin D1) ( F ) is presented relative to the expression of control gene HPRT . Statistical analysis was performed on four independent repeats using Student’s t -test. *, P- value <0.05; **, P <0.01. The experiments were repeated six times.

Article Snippet: AG01523 cells were transiently transfected with 20 nM siRNA of Trilencer-27 G3BP siRNA (#SR, OriGene Technologies, U.S.A.) or 20 nM scrambled negative control siRNA (#SR30004, OriGene Technologies, U.S.A.), using SilentFect reagent (Bio-Rad), and incubated for 72 to 96 h at 37°C in a CO 2 incubator.

Techniques: Expressing, Control

Journal: Bioscience Reports

Article Title: G3BP1 is a SWI/SNF-bound regulator of transcription that modulates activation of STATs

doi: 10.1042/BSR20250290

Figure Lengend Snippet: ( A ) G3BP1 was knocked down by siRNA, or not, in AG01523 fibroblasts. Cells were then grown in medium containing 1% FBS and increasing concentrations of PDGF-BB. The amount of DNA and, therefore, the proliferation rate was determined by measuring absorption of fluorescently labeled DNA-intercalating dye using the CyQuant assay. The experiments were repeated three times. ( B ) Schematic illustration of the findings of the study. G3BP1 acts as a co-activator of PDGF-BB-induced activation of STAT3 and as a co-repressor of the transcription of cyclin D1 and STAT1 due to its association with the SWI/SNF chromatin remodeling complex.

Article Snippet: AG01523 cells were transiently transfected with 20 nM siRNA of Trilencer-27 G3BP siRNA (#SR, OriGene Technologies, U.S.A.) or 20 nM scrambled negative control siRNA (#SR30004, OriGene Technologies, U.S.A.), using SilentFect reagent (Bio-Rad), and incubated for 72 to 96 h at 37°C in a CO 2 incubator.

Techniques: Labeling, CyQUANT Assay, Activation Assay