Journal: Human Molecular Genetics

Article Title: PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

doi: 10.1093/hmg/ddae051

Figure Lengend Snippet: Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in liver. (A and B) Western blot analysis of PAH, ubiquitin and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in liver samples from 12-week-old mice. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Statistical analysis performed by unpaired t test ( * P < 0.05, ** P < 0.01, ( *** P < 0.001, **** P < 0.0001, ns, not significant). Data are presented as mean ± SEM.

Article Snippet: For the analysis of total proteins , membranes were blocked for 1 h with 5% nonfat milk, in 0.1% TBS-tween and incubated overnight with the corresponding primary antibody: PAH (1:1000, Santa Cruz Biotechnology, sc-271 258), Ubiquitin (1:1000, Santa Cruz Biotechnology, sc-8017), Hsp70 (1:1000, Novus Biological, NB110-61582), DNAJC12 (1/1000, Abcam, ab167425), LC3B (1:1000, Cell Signaling Technology, #2775), LAMP1 (1:1000, Cell Signaling Technology, #3243) and p62/SQSTM1 (1:5000, Novus biologicals, H00008878-M01).

Techniques: Variant Assay, Western Blot, Ubiquitin Proteomics, Control

Journal: Human Molecular Genetics

Article Title: PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

doi: 10.1093/hmg/ddae051

Figure Lengend Snippet: Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in HepG2 cells. (A and B) Representative western blot analysis of PAH and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in HepG2 cells. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Data are presented as mean ± SD of two independent experiment. (D) PAH levels detected by western blot in HepG2 cells 72 h after transfection with 2 μg of the plasmid coding for DNAJC12. GAPDH was used as loading controls.

Article Snippet: For the analysis of total proteins , membranes were blocked for 1 h with 5% nonfat milk, in 0.1% TBS-tween and incubated overnight with the corresponding primary antibody: PAH (1:1000, Santa Cruz Biotechnology, sc-271 258), Ubiquitin (1:1000, Santa Cruz Biotechnology, sc-8017), Hsp70 (1:1000, Novus Biological, NB110-61582), DNAJC12 (1/1000, Abcam, ab167425), LC3B (1:1000, Cell Signaling Technology, #2775), LAMP1 (1:1000, Cell Signaling Technology, #3243) and p62/SQSTM1 (1:5000, Novus biologicals, H00008878-M01).

Techniques: Variant Assay, Western Blot, Control, Transfection, Plasmid Preparation

Journal: eLife

Article Title: Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome

doi: 10.7554/eLife.49138

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCMV6-MYC-DDK-HSP70-1A (HSPA1A) , OriGene , RC200270 , -.

Techniques: Recombinant, Software

Journal: International journal of molecular sciences

Article Title: Extracellular Heat Shock Protein 70 Increases the Glucocorticoid Receptor and Dual-Specificity Phosphatase 1 via Toll-like Receptor 4 and Attenuates Inflammation in Airway Epithelial Cells.

doi: 10.3390/ijms241411700

Figure Lengend Snippet: Figure 1. Effect of HSP70 overexpression on the expression of GR. (a) Representative Western blots of HSP70 transfection and the image analysis in NuLi-1. Alpha-tubulin served as the house-keeping control protein. Transfection of plasmid HSP70 induced iHSP70 expression in NuLi-1 cells. (b) GR mRNA (middle) and protein (right) expression was significantly reduced in cells overexpressing iHSP70. Bars show mean ± S.E.M. of three independent experiments. p-values are indicated as follows: * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

Article Snippet: Recombinant human TGFβ1 (#240-B) and recombinant human HSP70 (eHSP70; #AP-100) were purchased from R&D Systems (Abingdon, UK).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Exacerbation of mitochondrial fission in human CD34 + cells halts erythropoiesis and hemoglobin biosynthesis

doi: 10.1101/2020.07.31.230961

Figure Lengend Snippet: Total protein lysates from FIS1 OX and pWPI control cells at D8, D10, D13 and D16 of erythroid differentiation were examined by Western blot. (A) Protein levels of Procaspase 9 and cleaved active forms P37 and P35. This is for intrinsic apoptosis. HSP70 were used as housekeeping control. Band densitometry for p37, as a percentage of total caspase 9, was plotted. Values are +/− SEM. n =3. Statistical analysis, ANOVA followed by Bonferroni post hoc test. (B) Protein levels of Procaspase 8 and cleaved active forms P42/P43. This is for extrinsic apoptosis. HSP70 were used as housekeeping control. (C) Protein levels of Procaspase 3 and cleaved active form P17. This is a general apoptotic effector. HSP70 were used as housekeeping control. Band densitometry for p17, as a percentage of total caspase 3, was plotted. Values are +/− SEM. n =3. Statistical analysis, ANOVA followed by Bonferroni post hoc test (*** p <0.001).

Article Snippet: Total proteins were run on SDS-PAGE, transferred to PVDF membranes and blotted with the antibodies against FIS1 (ab71498, Abcam), MFN1 (ab104274,Abcam), ß-Actin (ab8227, Abcam), Caspase-3 (9665, Cell Signaling), Caspase-8 (sc-5263, Santa Cruz), Caspase-9 (sc-8355, Santa Cruz), HSP70 (TA309356, Origene) and Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam).

Techniques: Control, Western Blot