Fig. 3: High-throughput microscopy and genome editing as strategies to validate rAbs. | Communications Biology

Fig. 3: High-throughput microscopy and genome editing as strategies to validate rAbs.

From: High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics

Fig. 3

a Schematic representation of the criteria used to validate the antibodies by high-throughput microscopy. The number of antibodies that matches each criteria step is represented in the box. b Volcano plot representing the fluorescence intensity difference obtained from the comparison of two rAbs dilutions. Each dot represents one antibody tested. The x-axis describes the difference in the antibody fluorescence intensity at two different dilutions (1:500–1:100), and the y-axis describes the log of the desired False Discovery Rate (Q). Using GraphPad prism, the two-stage step-up method of Benjamini et al.13 was used to perform the analysis. The antibodies with Q higher than 2% were considered significant. c Representative image of Neuro2A cells probed with anti-phospho T500 PKCβ (AVG451P-G10) antibody at a dilution of 1:500 and 1:100 (green), and DAPI (blue). Images were acquired using the InCell microscope (×40) and fluorescence intensity measured by Cell Profiler software. Representative images with the antibody staining in green and DAPI in blue are shown. Volcano plot, each dot represents one cell. Lines are mean ± SD of two experiments, number of counted cells are indicated. RFU indicates the integrated fluorescence intensity units. ****p value < 0.0001; t-test. d Neuro2A and Neuro2A PKCβ-KO cells were probed with anti-phospho T500 PKCβ (AVG451P-G10) and anti-phospho T642 PKCβ (AVG453P-H11) antibodies (green) and DAPI (blue). Images were acquired and analyzed as previously described. ****p value < 0.0001; t test. e Validation of phosphospecific antibodies against PKC substrates. Neuro2A and Neuro2A PKCα-KO cells were probed with anti-phospho S159/S163 MARCKS (AVG 457-G10); anti-phospho pS81 Ppp2r5d (AVG709P1-E11) antibodies after PMA treatment (100 nM) for 30 min; anti-phospho S363 MOR (AVG710P-B7) antibody after PMA treatment (100 nM) for 60 min; anti-phospho S375/T376 MOR (AVG713-B5) antibody after PMA treatment (100 nM) for 30 s. Images were acquired and analyzed as previously described. ****p value < 0.0001; t test.

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