For immunoprecipitation experiments, cells were lysed in 140 mM NaCl, 10 mM Tris pH 7.6, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, protease and phosphatase inhibitors. Cells were incubated on ice for 30′, then centrifuged for 20′ at 4 °C, at 13000 rpm. Thereafter, supernatant (1–2 mg) underwent immunoprecipitation as described33 (link), by using 4 μg of each antibody or control IgGs (mouse IgGs were from Santa Cruz, cat. #sc-2025; rabbit IgGs were from Merck, #cat. I8140). To avoid IgGs detection, either ImmunoCruz IP/WB Optima B, C, F, E (Santa Cruz Biotechnology; cat. #sc-45039, cat. #sc-45040, cat. #sc-45043, cat. #sc-45042, respectively) or the VeriBlot reagent (Abcam; #cat. ab131366 and #ab131368) were used in western blot, after the immunoprecipitation procedure, according to the manufacturer’s instructions.
Mouse iggs
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom
Mouse IgGs are antibody proteins derived from mice. They are used as research tools in various laboratory applications.
Automatically generated - may contain errors
3 protocols using mouse iggs
1
Immunoprecipitation Workflow Optimization
2
Immunoprecipitation and Kinase Assay Protocol
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For immunoprecipitation, soluble cell extracts or HNE (CIL Biotech) were incubated with Magnetic protein G or protein A beads (Invitrogen) bound to specific Abs in binding buffer (50 mM Hepes [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 [NP-40], a protease inhibitor cocktail [cOmplete Mini, Roche Diagnostic] and phosphatase inhibitors [2 mM Na3VO4, 30 mM Na4P2O7, 25 mM NaF]). The Abs used were: DYRK1A Ab-C1 (Abnova H00001859-M01); DYRK1A Ab-C2 (Abcam ab69811); DYRK1A Ab-N (Sigma D1694); FLAG (Sigma, F1804); HA (BioLegend, 901501); RNF169 (Bethyl Laboratories, A304-097A); or control immunoglobulin G (rabbit IgGs, Cell Signaling, #2729S; mouse IgGs, Santa Cruz, #sc-2025). The beads were then washed three times with binding buffer, and once with the same buffer without NP-40. The samples were analyzed in Western blots (WBs) or used for IVK assays. Details on the preparation of the cell extracts and WB analysis are provided in the Supplementary Methods.
3
Quantifying Macrophage Polarization Markers
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Ti substrates) at a density of 9.5 x 10 4 cells/cm 2 using complete culture media. To evaluate the polarization toward M1 or M2-like phenotypes, the specific markers, CCR7 and CD206, were used. After 24 h of culture, the surfaces were washed with PBS and fixed with 10 % buffered formalin. The samples were stained with primary antibody anti-CCR7 (1:250, rabbit IgG, abcam, UK) or anti-CD206 (1:50, mouse IgGS, SantaCruz Biotechnology) and secondary antibodies, donkey anti-rabbit IgG Alexa Fluor 488 (1:200; ThermoFisher Scientific) and rabbit anti-mouse IgG Alexa Fluor 488 (1:200; ThermoFisher Scientific), respectively. Nuclei and cytoskeleton were stained using DAPI and Phalloidin-TRITC, as previously described. Samples were visualized using an inverted fluorescence microscope (Axio Observer, Zeiss). The cell aspect ratio was calculated from the tile images acquired for each sample. The quantification was performed with the particle analysis tool of Fiji (Image J) using the aspect ratio subset and after establishing a threshold. CCR7 expression was quantified by applying a threshold to discard background pixels, and then the area of green pixels, corresponding to CCR7 positively stained cells, was divided by the number of cells. The experiments were repeated three times with duplicates of each condition.
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