Journal: Molecular Medicine Reports

Article Title: Expression and function of Toll-like receptors in peripheral blood mononuclear cells in patients with ankylosing spondylitis

doi: 10.3892/mmr.2019.10631

Figure Lengend Snippet: TNF-α mediates TLR expression and NF-κB p65 signaling in PBMCs. (A) Western blotting was performed to detect the protein levels of TLR3, TLR4, TLR5, p65 and p-p65. Protein expression of (B) TLR3, (C) TLR4, (D) TLR5 and (E) p-p65/p65 presented as bar diagrams. β-actin was used as an internal control. *P<0.05, **P<0.01 vs. control. NF-κB, nuclear factor-κB; p-, phosphorylated; PBMC, peripheral blood mononuclear cell; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α.

Article Snippet: The membranes were blocked in 5% dried skimmed milk in TBS buffer at 37°C for 1 h and incubated overnight at 4°C with antibodies against TLR3 (1:700; ab62566, Abcam), TLR4 (1:600; ab13556, Abcam), TLR5 (1:800; ab62460, Abcam), p65 (1:1,000; ab32536, Abcam), phosphorylated (p)-p65 (1:600; ab86299, Abcam) and β-actin (1:1,000; MAB8969, R&D Systems, Inc.).

Techniques: Expressing, Western Blot, Control

Journal: PLoS Biology

Article Title: Small molecule Y-320 stimulates ribosome biogenesis, protein synthesis, and aminoglycoside-induced premature termination codon readthrough

doi: 10.1371/journal.pbio.3001221

Figure Lengend Snippet: (A) p53 PTC readthrough in HDQ-P1 cells exposed to the indicated compounds for 48 h. Shown are pseudo blots of chemiluminescence of bound p53 and vinculin antibodies. The area under the FL-p53 peaks was first normalized to the vinculin loading control and then divided to that of G418 to provide lane-to-lane comparison. These numbers are displayed under the lanes. The MW ladder in kDa is shown at the left. (B) p53 PTC readthrough in H1299-R213X cells exposed to the indicated compounds for 24 h and imaged as in panel A. (C) Dystrophin PTC readthrough. WT and DMD patient-derived myoblasts were exposed to the indicated compounds in differentiating medium for 5 days. The proteins were separated using high-molecular weight capillaries. Shown are pseudo blots of bound dystrophin and GAPDH antibodies. The area under the dystrophin peaks was first normalized to the GAPDH loading control and then divided to that of G418 to provide lane-to-lane comparison. These numbers are displayed under the lanes. The amount of WT protein run in the capillary was 5% of that of DMD cells. (D) Collagen XVII PTC readthrough. Junctional Epidermolysis Bullosa patient-derived keratinocytes (JEB01) were exposed to the indicated concentrations of G418 and Y-320 for 72 h. COL17A1 protein levels were determined by traditional western blotting. The blots were scanned and COL17A1 band intensities were measured and normalized to the beta-actin loading control using ImageJ. The values are expressed relative to 30 μM G418 conditions to provide lane-to-lane comparison and are displayed under the lanes. The amount of HaCat protein run in the capillary was 10% of that of JEB cells. Dashed lines indicate different capillaries or blots. Uncropped images of automated capillary electrophoresis western analyses are provided in . DMD, Duchenne Muscular Dystrophy; FL-p53, full-length p53; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; PTC, premature termination codon; TR-p53, truncated p53; WT, wild-type.

Article Snippet: Mouse anti-p53 antibody (1:400, DO-1, Santa Cruz sc-126), mouse anti-vinculin antibody (1:600 for HDQ-P1 and 1:1,000 for H1299-R213X and Tsc2 −/− cells, R&D Systems MAB6896), rabbit anti-phospho-p70 S6K (Thr389) (1:50, Cell Signaling Technology 9205), rabbit anti-phospho-4EBP1 (Thr37/46) (1:200, Cell Signaling Technology 9459), rabbit anti-Collagen XVII (1:25, Abcam ab184996), rabbit anti-beta-actin (1:10,000, Novus Biologicals), rabbit anti-Dystrophin (1:400, Abcam ab15277), rabbit anti-eIF4G (1:100, Cell Signaling Technology 2498), and rabbit anti-phospho-eIF4B (Ser422) (1:150, Cell Signaling Technology 3591) were used.

Techniques: Control, Comparison, Derivative Assay, High Molecular Weight, Western Blot, Electrophoresis, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: MiR-7-5p Is Involved in Ferroptosis Signaling and Radioresistance Thru the Generation of ROS in Radioresistant HeLa and SAS Cell Lines

doi: 10.3390/ijms22158300

Figure Lengend Snippet: Expression of ALOXs in CRR cells. ALOX5 , 12, and 15 expressions in parent and CRR cells were detected using qPCR ( A – C ) and western blotting ( D ). A: Gene expression of ALOX5 . ( B ) Gene expression of ALOX12 . C: Gene expression of ALOX15 . **: p < 0.01 using Student’s t -test. Each qPCR was performed 3 times. Gene expressions of ALOX5 , 12 , and 15 were all significantly down-regulated in CRR cells. ( D ) Western blotting of ALOX5 , 12 , and 15 . The table shows the expression level of each ALOX s in CRR cells. After correcting the intensity of each band by the expression of β-actin, the value is shown with the parent strain as 1. The protein expressions of ALOX5 and 12 were down-regulated in CRR cells. However, ALOX15 was not down-regulated in protein level in SAS CRR cells.

Article Snippet: Anti-β–actin antibody (NB100-56874; Novus Biologicals LLC, Centennial, CO, USA, dilution factor: 1:1000) was used as a loading control.

Techniques: Expressing, Western Blot, Gene Expression