Journal: International Journal of Clinical and Experimental Medicine

Article Title: Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites

doi:

Figure Lengend Snippet: Primer pairs for target and housekeeping genes used in qRT-PCR assays

Article Snippet: Cell lysates were resolved by standard SDS-PAGE and electroblotted onto a PVDF membrane and incubated with rabbit anti human integrin β1 (Abcam, Cambridge, MA) and rabbit anti human p130Cas antibodies (Cell Signaling Technology, Inc., Danvers, MA) and with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugate antibody (CWbiotech Inc., Beijing, China).

Techniques:

Journal: International Journal of Clinical and Experimental Medicine

Article Title: Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites

doi:

Figure Lengend Snippet: Effects of mechanical stress on target gene expressions in stretched upper back and medial arm skin fibroblasts. At each time points of mechanical loads, individual cultures of fibroblasts were collected and assayed for relative levels of gene expression of integrin β1 (Panel A), p130Cas (Panel B), TGF β1 (Panel C) and type I collagen (Panel D). Data are shown as the mean ± SD based on triplicate experiments. * and †P<0.05 versus medial arm fibroblasts stressed for 36 and 48 hours, respectively.

Article Snippet: Cell lysates were resolved by standard SDS-PAGE and electroblotted onto a PVDF membrane and incubated with rabbit anti human integrin β1 (Abcam, Cambridge, MA) and rabbit anti human p130Cas antibodies (Cell Signaling Technology, Inc., Danvers, MA) and with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugate antibody (CWbiotech Inc., Beijing, China).

Techniques: Gene Expression

Journal: International Journal of Clinical and Experimental Medicine

Article Title: Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites

doi:

Figure Lengend Snippet: Effects of mechanical stress on target protein productions in stretched upper back and medial arm fibroblasts. At each time point following mechanical loads, each culture of fibroblasts was collected and used for Western-blot detection of integrin β1 and p130Cas (Panel A) and afterward semi-quantified and normalized to that of internal reference of GAPDH respectively (Panel B, C); or the conditioned supernatant from each group for ELISA assay to detect protein contents of TGF β1 and type I collagen (Panel D and Panel E, respectively). Data are shown as the mean ± SD based on triplicate experiments. * and †P<0.05 versus medial arm fibroblasts stressed for 36 and 48 hours, respectively. M: Medial side of upper arm; S: Scapular part of upper back skin.

Article Snippet: Cell lysates were resolved by standard SDS-PAGE and electroblotted onto a PVDF membrane and incubated with rabbit anti human integrin β1 (Abcam, Cambridge, MA) and rabbit anti human p130Cas antibodies (Cell Signaling Technology, Inc., Danvers, MA) and with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugate antibody (CWbiotech Inc., Beijing, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Evolution of CRISPR-associated Endonucleases as Inferred from Resurrected Proteins

doi: 10.1101/2022.03.30.485982

Figure Lengend Snippet: (a) Phylogenetic chronogram of Cas9 endonucleases. Fifty-nine sequences were chosen from two phyla, Firmicutes and Actinobacteria, with two classes, Bacilli and Clostridia, belonging to Firmicutes. Identification codes of all sequence can be found in the Supplementary Information. Divergence times were estimated using Bayesian inference and information from the Time Tree of Life. Internal nodes from Firmicutes Common Ancestor (FCA), Bacilli Common Ancestor (BCA), Streptococci Common Ancestor (SCA), Pyogenic Common Ancestor (PCA) and Pyogenes-Dysgalactiae Common Ancestor (PDCA) were selected for testing. (b) Superposition of structural prediction of FCA Cas9 using AlphaFold2 (blue) with x-Ray structure of SpCas9 with guide RNA and target DNA (PDB:4oo8, red). (c) Isolated HNH domains of SpCas9 (red) and FCA anCas (blue) from whole structure coordinates. (d) Superposition of structural prediction of PDCA anCas with guide RNA and target DNA using AlphaFold2 (purple) with x-Ray structure of SpCas9 (PDB:4oo8, red). (e) pLDDT values for all anCas as estimated from AlphaFold2 prediction.

Article Snippet: Anti-Cas9 rabbit antibody (Rockland, 600-401-GK0) was diluted 1:25000 in 1% BSA blocking solution and plates were incubated for 2 hours at room temperature.

Techniques: Sequencing, Structural Proteomics, Isolation

Journal: bioRxiv

Article Title: Evolution of CRISPR-associated Endonucleases as Inferred from Resurrected Proteins

doi: 10.1101/2022.03.30.485982

Figure Lengend Snippet: (a) In vitro cleavage assay on a supercoiled DNA substrate of anCas and SpCas9 using sgRNAs from different species. FCA, BCA anCas and SpCas9 are shown. (b) Quantification of in vitro cleavage for all anCas and SpCas9 using the different sgRNAs. (c) In vitro cleavage assay on an 85 nt ssDNA fragment at different incubation times for FCA, BCA anCas and SpCas9. (d) In vitro cleavage assay on a 60 nt ssRNA at different incubation times for FCA, BCA anCas and SpCas9. (e) Quantification of fraction cleavage of ssDNA at different times and exponential fits for determination of kinetics parameters. In both (c) and (d), the control lane is the same for the three proteins. (f) Quantification of fraction cleavage of ssRNA at different times and exponential fits for determination of kinetics parameters. All kinetics parameters are summarized in . (g) Results from ELISA test of Anti-Cas9 rabbit antibody against SpCas9, FCA anCas, BCA anCas and BSA, used as control. Results are reported as average and S.D from three independent experiments.

Article Snippet: Anti-Cas9 rabbit antibody (Rockland, 600-401-GK0) was diluted 1:25000 in 1% BSA blocking solution and plates were incubated for 2 hours at room temperature.

Techniques: In Vitro, Cleavage Assay, Incubation, Control, Enzyme-linked Immunosorbent Assay