P2X4R, BDNF and Iba1 detection using western blotting.

Western blot of P2X4R and Iba1 proteins (A), and α-tubulin, BDNF and its isoforms in SIM-A9 cell lysates (B) collected from different passages. In each blot, the series of bands on the left shows the protein ladder. For P2X4R and Iba1 (A), cell lysates collected from P4 and P5, at 30, 40, and 50μg/lane total protein were used to detect P2X4R (green, 43kD) and Iba1 (green, 17kD) on the 800 nm channel. The signals at about 25 kD are due to the non-specific reactivity of secondary antibody. For α-tubulin and BDNF (B), cell lysates at 30 and 40 μg/lane total protein were electrophoresed to identify α-tubulin (50 kD), BDNF monomer (14 kD), BDNF dimer (28 kD), and pro-BDNF (37 kD) on the 700 nm and 800 nm channels, respectively. The blots were scanned using an Odyssey imager at intensity setting 5 and processed using ImageStudio 5.2 software. Each sample was run in triplicates (A) or duplicates (B). C-F. Densitometry analysis of P2X4R, Iba1, BDNF, and BDNF dimer bands was performed using ImageStudio 5.2 software. Signal intensities of P2X4R (C) and Iba1 (D) bands were normalized for protein loading. Signal intensities of BDNF (E) and BDNF dimers (F) were first normalized to α-tubulin and then to the amount of protein loaded. Statistical analysis between P4 and P5 was performed by unpaired t-test and Welch’s correction using GraphPad Prism 8.1.2. Asterisks indicate statistically significant differences (* p<0.05). Blots were merged and cropped for the clarity and conciseness of the presentation. Full-length blots are presented in S3A and S3B Fig, raw blots A and B.