Journal: Scientific Reports

Article Title: Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

doi: 10.1038/s41598-020-75375-0

Figure Lengend Snippet: Protein microarray data indicate that phosphorylation of S298 in maCx35 may act as a functional switch. ( a ) Membrane topology of connexins. N- and C-terminal tails (NT and CT, respectively) and the cytoplasmic loop (CL) reside inside the cytoplasm. The two extracellular loops (EL) are also indicated. ( b ) Sequence of the C-terminal end of maCx35/mmCx36, as indicated by the box in ( a ). Sequence identity (*) is high for mmCx36 and maCx35 at the C-terminal end. The blue box indicates a PKA/CaMKII consensus site at position S315 and S298 for mmCx36 and maCx35, respectively. The last four amino acids represent a PDZ binding domain, present in both proteins. ( c ) Microarray layout (A-D); the respective baits (all fused to GST) are found below. An array probed with Alexa Fluor 555 anti-GST antibody served as positive control. Peptides fused to biotin containing the C-terminal tail of maCx35 (35 WT), phosphorylated maCx35 (35 S298 phos), and a truncated version (35 S298 ter) were used as probes and showed differential results, marked by rounded squares. 14–3–3 proteins (red) and NHERF2 aa265-288 (blue D7) showed an increased binding to the 35 S298 phospho probe compared to 35 WT. In contrast, PDZK1, PDZ10 of MUPP1, and nNOS (blue) decreased their binding to the 35 S298 phospho probe. The truncated 35 S298 ter probe showed no binding interactions.

Article Snippet: Purified GST fusion proteins (listed in Fig. c and Supplementary Fig. ) were spotted onto nitrocellulose-coated glass slides with an Aushon 2470 microarray robot; each protein was spotted in duplicate.

Techniques: Microarray, Functional Assay, Sequencing, Binding Assay, Positive Control

Journal: Journal of visualized experiments : JoVE

Article Title: Analysis of Histone Antibody Specificity with Peptide Microarrays

doi: 10.3791/55912

Figure Lengend Snippet: Materials

Article Snippet: contact microarray printer , Aushon , 2470 , Aushon 2470 Microarray Printer.

Techniques: Microscopy, Microarray, Blocking Assay, Plasmid Preparation, Hybridization, Peptide Microarray, Modification, High Throughput Screening Assay

Journal: Journal of visualized experiments : JoVE

Article Title: Analysis of Histone Antibody Specificity with Peptide Microarrays

doi: 10.3791/55912

Figure Lengend Snippet: A screen shot of the ArrayNinja design module is shown in the dotted line. The control panel (top) shows all of the parameters that can be altered on the microarray printer. As these parameters are adjusted, the cartoon image of the slide layout (bottom left) updates in real time. After the layout is set, the user can mouse over individual spots to enter unique feature identifiers. ArrayNinja constructs from this user input a map of the position of each feature in the source plate(s) (bottom right) needed to fabricate a specified microarray slide layout.

Article Snippet: contact microarray printer , Aushon , 2470 , Aushon 2470 Microarray Printer.

Techniques: Microarray, Construct

Journal: Journal of visualized experiments : JoVE

Article Title: Analysis of Histone Antibody Specificity with Peptide Microarrays

doi: 10.3791/55912

Figure Lengend Snippet: (A) Histone peptide microarray fabrication on streptavidin-coated microscope slides using a contact microarray printer. (B) Microarrays fabricated with 3 subarrays of a 48 × 48 grid of peptide features. Separation of (C) 3 subarrays with a hydrophobic wax pen, (D) 2 subarrays with a silicon adhesive, and (E) 48 subarrays with a wax imprint. All microarrays shown are fabricated using 25 × 75 mm microscope slides.

Article Snippet: contact microarray printer , Aushon , 2470 , Aushon 2470 Microarray Printer.

Techniques: Peptide Microarray, Microscopy, Microarray