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: EJNMMI Research

Article Title: Radiosensitization of NET cells by HSP90 inhibitor ganetespib is mediated through pleiotropic stress responses

doi: 10.1186/s13550-025-01346-z

Figure Lengend Snippet: Efficacy of combination treatment of ganetespib with EBRT and PRRT. (A) Time-dependent induction of HSP70 protein expression after start of ganetespib treatment (10 or 25 nM) for GOT1 (left) and BON1-SSTR2 (right) cells. β-actin was used as loading control. (B) Viability assay for GOT1 (left) and BON1-SSTR2 (right) cells of a concentration range of ganetespib as monotherapy, or combined with 2 Gy EBRT or 1 MBq/mL PRRT 7 days post treatment initiation. Values are expressed as the percentage viability and normalized to their respective viability without ganetespib (0 nM). Data points represent the mean of 3 independent biological replicates with technical triplicates. Below the graphs, the viability of PRRT or EBRT monotherapy is depicted. (C) Assessment of cell death for GOT1 (left) and BON1-SSTR2 (right) cells after ganetespib (0, 10 or 25 nM) as monotherapy, or combined with 2 Gy EBRT or 1 MBq/mL PRRT 3 days post treatment initiation. Values are expressed as the percentage of living cells and normalized to their respective control condition (without ganetespib, 0 nM) for each curve. Data points represent the mean of 3 independent biological replicates. Below the graphs, the relative percentage of live cells of PRRT or EBRT monotherapy is depicted. All error bars represent the SEM. Asterisks indicate the level of significance of the difference for each corresponding point with control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Article Snippet: Membranes were blocked in PBS + 0.05% Tween (Sigma-Aldrich) + 3% skimmed milk (Merck Millipore) and incubated with primary antibodies rat anti-HSP70 (Cell Signaling Technology, 4873 S, 1:1000) and mouse anti-actin (Millipore, LV1547855, 1:500.000) at 4 °C overnight.

Techniques: Expressing, Control, Viability Assay, Concentration Assay

Journal: Nature Communications

Article Title: CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion

doi: 10.1038/s41467-021-22927-1

Figure Lengend Snippet: a Scheme depicting a generic CopyCatcher element. Light blue boxes: exons of a targeted gene on the donor chromosome; dark blue boxes: exons on the receiver chromosome; black lines: genomic DNA; red box: splice acceptor site (SA); yellow box: T2A self-cleavage peptide; light red arrow: DsRed reporter; dark purple arrow: gRNA; light blue arrow: selection marker mCerulean ; red hourglass marks: point mutations at or near initiator ATG codons of endogenous genes; short black line: Cas9/gRNA cleavage site; light blue circle and dark lines: Cas9/gRNA complex. Scissors in ( a ) denote insertion of cargo cassettes into DSBs on homologous chromosomes including SA, T2A, gRNA, and selection marker mCerulean . b – d Distinct outcomes of three DSB repair mechanisms followed by Cas9/gRNA cleavage of the homologous “receiver” chromosome. Slashes on the second exons in ( b )–( d ) indicate loss of function on the marked chromosomes. Labels indicate resulting phenotypes. e, f Mosaic clones of somatic gene conversion (SGC) in two CopyCatcher lines. Photographs show the phenotypes and fluorescence patterns of two CopyCatcher elements inserted into the intron of white and ple loci in flies without (( e ) w [3XP3-CC] , ( f ) ple [CC] ) or with (( e ) w [ATG-,3XP3-CC] , ( f ) ple [ATG-,CC] ) associated ATG – point mutations, or F1 mosaics resulting from Cas9-mediated copying (( e ) w [ATG-,3XP3-CC] / w +; Cas9/+, ( f ) ple [ATG-,CC] /Cas9). SGC clones generated by ple [ATG-,CC] are outlined with white dotted lines. Rightmost panels in ( e ) and ( f ) are higher magnification views of areas delineated by white boxes in the lower magnification views immediately to their left. At least five independent flies were imaged and observed in ( e ) and ( f ) with similar results. Scale bars stand for 150 pixels in ( e ) and ( f ).

Article Snippet: The SA-T2A sequence was amplified from pBS-KS-attB2-SA-T2A-3XGal80-Hsp70 series plasmids (Addgene 62951 and 62952) , gRNAs-expressing cassette targeting to the first intron of yellow , white , and ple were assembled following online published protocols (CRISPR fly Design - http://www.crisprflydesign.org/ ) and mCerulean selection marker was amplified from Addgene 27795 plasmid.

Techniques: Selection, Marker, Clone Assay, Fluorescence, Generated

Journal: Oncotarget

Article Title: The sphingosine 1-phosphate receptor 2 is shed in exosomes from breast cancer cells and is N-terminally processed to a short constitutively active form that promotes extracellular signal regulated kinase activation and DNA synthesis in fibroblasts

doi: 10.18632/oncotarget.25658

Figure Lengend Snippet: (A) Western blot with anti-GFP or anti-mCherry antibodies showing the over-expression of GFP-hsp70 and mCherry-tgs101 in vector (V) or plasmid transfected MDA-MB-231 cells for 24 or 48 h. (B) Western blot with anti-GFP or anti-mCherry or anti-HA antibodies showing the presence of GFP-hsp70, mCherry-tsg101 or HA tagged S1P 2 (Mr = 40 kDa) in transfected MDA-MB-231 cell lysate (CL) and acid precipitates (PPT) of CM. (C) Western blot with anti-hsp70, anti-CD63 and anti-S1P 2 antibodies showing the presence of GFP-hsp70, CD63 or S1P 2 in MDA-MB-231 cell lysates (CL) and isolated exosomes (EXO) from CM. (D) Immunofluorescence image of MDA-MB-231 cells stained with anti-CD63/TRITC (red) secondary and anti-S1P 2 /FITC (green) secondary antibodies showing co-localisation (yellow) of CD63 and S1P 2 in large vesicles typical of MVBs. (E) Electron micrograph of immunogold staining with anti-CD63 [attached to secondary goat anti-mouse IgG-immune-gold particles (15 nm)] and anti-S1P 2 antibodies [attached to secondary goat anti-rabbit IgG-immune-gold particles (10 nm)] in exosomes isolated from CM of MDA-MB-231 cells. Control represents exosomes that have not been incubated with primary antibody. Results are representative of 3 independent experiments.

Article Snippet: Plasmid constructs for pEGFP hsp70 (# 15215) and pEF6. mCherry-tsg101 (# 38318) were from Addgene (Cambridge, MA, USA) and for HA-tagged S1P 2 (EDG050TN000) from UMR cDNA Resource Center (Bloomsberg, USA).

Techniques: Western Blot, Over Expression, Plasmid Preparation, Transfection, Isolation, Immunofluorescence, Staining, Control, Incubation

Journal: Oncotarget

Article Title: The sphingosine 1-phosphate receptor 2 is shed in exosomes from breast cancer cells and is N-terminally processed to a short constitutively active form that promotes extracellular signal regulated kinase activation and DNA synthesis in fibroblasts

doi: 10.18632/oncotarget.25658

Figure Lengend Snippet: MEFs quiescent for 24 hr were treated with NCM or CM from MDA-MB-231 cells for 10 min. MEFs were pre-treated with and without JTE-013 (10 μM) or CYM5520 (10, 25 μM) for 15 min prior to addition of NCM or CM. (A) Western blot showing the lack of effect of CYM5520 alone on ERK-1/2 activation in MEFs. (B) Western blot showing the uptake into MEFs of the short S1P 2 (Mr = 36 kDa) form and the activation of ERK-1/2 in response to CM isolated from MDA-MB-231 cells. (A) and (B) Blots were re-probed with anti-actin antibody to ensure equal protein loading. (C) Immunofluorescence images of control, CM-stimulated and CYM5520 (10 μM)-stimulated MEFs co-stained with anti-actin (TRITC secondary antibody, red) and anti-S1P 2 (FITC secondary antibody, green) antibodies. (D) Fluorescence image of MEFs showing uptake of GFP-hsp70 by detection of GFP and co-stained with anti-actin antibody using a Texas Red conjugated secondary antibody or DAPI. Results are representative of 3 independent experiments.

Article Snippet: Plasmid constructs for pEGFP hsp70 (# 15215) and pEF6. mCherry-tsg101 (# 38318) were from Addgene (Cambridge, MA, USA) and for HA-tagged S1P 2 (EDG050TN000) from UMR cDNA Resource Center (Bloomsberg, USA).

Techniques: Western Blot, Activation Assay, Isolation, Immunofluorescence, Control, Staining, Fluorescence