scrambled shrna control with tgfp  (OriGene)

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: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: TCF7 regulates pro−caspase−8 expression in T lymphocytes and is significantly reduced in COPD. ( A ) Immunofluorescence co−staining of control human lung tissue displaying separate channels for DAPI (blue), caspase−8 (green), TCF7 (red), and the merged image. Scale bar is 50 μm. ( B ) Immunofluorescence co−staining of COPD human lung tissue displaying separate channels for DAPI (blue), caspase−8 (green), TCF7 (red), and the merged image. Note the marked reduction in both TCF7 and caspase−8 signals compared to the control. Scale bar is 50 μm. ( C ) Representative Western blot images of TCF7 (50 kDa), pro−caspase−8 (55 kDa), and internal control β−tubulin (55 kDa) in wild type (WT) and TCF7 knockout (KO) Jurkat T cells. ( D ) Quantitative densitometric analysis of TCF7 protein levels comparing WT and KO groups. ( E ) Quantitative densitometric analysis of pro−caspase−8 protein levels comparing WT and KO groups. ( F ) Representative Western blot images of TCF7 and β−tubulin in primary T lymphocytes isolated from the peripheral blood of healthy donors (Control) and patients with COPD (Model). ( G ) Quantitative densitometric analysis of TCF7 protein levels in human primary T lymphocytes. ( H ) Representative Western blot images of TCF7 and β−tubulin protein levels in Jurkat T cells across four experimental conditions including Control, shRNA, shRNA plus TCF7 Rescue construct, and shRNA plus Empty Vector. ( I ) Quantitative densitometric analysis of TCF7 protein levels across the four experimental rescue groups. ( J ) Representative Western blot images of pro−caspase−8 and β−tubulin protein levels across the same four experimental conditions in Jurkat T cells. ( K ) Quantitative densitometric analysis of pro−caspase−8 protein levels across the four experimental rescue groups. Data in the bar charts are presented as mean ± SD ( n = 4 for primary human cells, n = 3 for cell line experiments). Statistical significance was assessed using Student’s t test with Welch’s correction where appropriate (* p < 0.05, *** p < 0.001, ns indicates not significant).

Article Snippet: Short hairpin RNA targeting human TCF7 (shRNA) and a scramble control shRNA were purchased from OriGene with Cat.No.TR30004.

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, Knock-Out, Isolation, shRNA, Construct, Plasmid Preparation