Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Journal of Biological Chemistry

Article Title: A Single-nucleotide Deletion Leads to Rapid Degradation ofTAP-1 mRNA in a Melanoma Cell Line

doi: 10.1074/jbc.m300954200

Figure Lengend Snippet: FIG. 1. Deficiency of surface HLA class I expression in mela- noma cell line SK-MEL-19 was due to the TAP-1 down-regula- tion. a, HLA class I expression in three melanoma cell lines, SK-MEL- 19, 1102, and 1195. Bold black lines depict the staining by PE- conjugated anti-human HLA-A, -B, and -C antibody in untreated cells; dotted lines represent the staining by PE-conjugated mouse IgG1 as isotype control; and red lines represent anti-HLA-A, -B, and -C antibody staining after stimulation with 1000 units/ml IFN- for 72 h. b, expres- sion of HLA class I heavy chain (MHC I), 2M, TAP-1, TAP-2, LMP-2, and LMP-7 in each cell line with or without IFN- induction (1000 units/ml for 72 h). Total RNA loading to each well was shown as 28 S rRNA (28S) and 18 S rRNA (18S). c, transfection with wild type TAP-1, but not vector alone, restored HLA class I expression in the SK-MEL-19 cells. SK-MEL-19 cells were transfected with either vector alone (top panels) or vector with TAP-1 cDNA insert (bottom panels). These stable clones from each group were stimulated with or without IFN- and analyzed for cell surface HLA-A, -B, and -C, as detailed in a.

Article Snippet: For induction of HLA class I expression, cells were cultured in medium supplemented with recombinant human IFN- (1000 units/ml; R&D Systems, Minneapolis, MN).

Techniques: Expressing, Staining, Control, Transfection, Plasmid Preparation, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: A Single-nucleotide Deletion Leads to Rapid Degradation ofTAP-1 mRNA in a Melanoma Cell Line

doi: 10.1074/jbc.m300954200

Figure Lengend Snippet: FIG. 2. Posttranscriptional mechanisms are responsible for poor accumulation of TAP-1 mRNA. a, a single-nucleotide polymorphism, adjacent to the first transcription start site (427), was identified at 446 in the bidirectional promoter shared by the TAP-1 and LMP-2 genes. The G 3 T change results in the loss of the AflIII restriction site. Southern blot hybridization was performed using AflIII and detected by a DNA probe that encompasses the downstream region of the polymorphism site. SK-MEL-19 cells showed one 5.6-kb band that represents homozygous T allele, as did the breast cancer cell line SK-BR-3, which has significant cell surface HLA class I surface expression (data not shown). HeLa cells, in contrast, are homozygous for the G allele. b, activities of T and G alleles of TAP-1 promoter (pTAP1/T and pTAP1/G, respectively) in SK-MEL-19 cells. The two allelic forms of TAP-1 promoter were cloned into pGL2-basic vector (basic) that did not contain any promoter or enhancer but encoded firefly luciferase. The pGL2-SV40 construct (SV40) that had both SV40 promoter and SV40 enhancer as well as the firefly luciferase reporter gene was used as positive control. After transfection, IFN- was added to the cell culture at 1000 units/ml. Cells were lysed 48 h after transfection, and luciferase expression was tested using a luminometer. Data shown are representative of at least five independent experiments. c, the TAP-1 gene was actively transcribed in SK-MEL-19 cells in the presence and absence of IFN- in nuclear run-on assay. Endogenous GAPDH expression was used as a positive control, and the pcDNA3.1/Hyg() vector was used as a negative control. The run-on experiments were repeated three times with similar results.

Article Snippet: For induction of HLA class I expression, cells were cultured in medium supplemented with recombinant human IFN- (1000 units/ml; R&D Systems, Minneapolis, MN).

Techniques: Southern Blot, Hybridization, Expressing, Clone Assay, Plasmid Preparation, Luciferase, Construct, Positive Control, Transfection, Cell Culture, Nuclear Run-on Assay, Negative Control

Journal: bioRxiv

Article Title: Csf1, a tunnel-like lipid-transfer protein, mediates lipid remodeling and underpins eukaryotic membrane resilience to high hydrostatic pressure and cold

doi: 10.1101/2025.11.20.689427

Figure Lengend Snippet: Whole-cell extracts, P13 and P100 membrane fractions prepared from cells were analyzed by Western blotting to detect marker proteins (Pma1 for the PM, Dpm1 for the ER membrane, and Pep12 for endosomes). WCE, whole cell extracts.

Article Snippet: Western blotting was performed for the whole cell extracts, P13 and P100 membrane fractions, as previously described, using an anti-HA monoclonal antibody (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan), anti-Pma1 polyclonal antibody ( Kato et al ., 2025 ), anti-Pep2 monoclonal antibody (2C3G4, ab113689, Abcam Inc., Waltham MA, USA), anti-dolichol phosphate mannose synthase (Dpm1) monoclonal antibody (5C5, A6429, ThermoFisher Scientific), and horseradish peroxidase-conjugated anti-mouse IgG antibody (GE Healthcare Life Sciences, Piscataway, NJ, USA).

Techniques: Membrane, Western Blot, Marker