Anti app clone 22c11

Proteins in cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes blocked with 4% nonfat milk in Tris-buffered saline containing 0.2% Tween 20. Blots were immunostained with anti-APP (clone 22C11, Millipore) and anti-tubulin (clone B-5-1-2, Sigma-Aldrich) monoclonal antibodies at 4 °C overnight. Blots were probed with secondary antibodies conjugated to horseradish peroxidase (Invitrogen). Specific bands were visualized with enhanced chemiluminescence (Pierce). Quantification was performed with ImageJ software.

A number of selected proteins that were detected as regulated in the analysis were further validated by immunoblotting (TIMP-3, TIMP-1, SPARC, MIF, APP). Supernatants from TIMP-3/HEK or HEK293 cells were loaded onto an acrylamide gel for electrophoresis. After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described^{65 (link)}), anti-TIMP-2 (generated as previously described^{66 (link)}), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) anti-actin (A5316, Sigma Aldrich).
Bands corresponding to each protein were quantified by using Multi Gauge software (Fujifilm) and normalized to the mean of the original non-normalized control values (HEK293 cells). A two-sided Student’s t-test was used to evaluate proteins statistically significantly regulated, with a p-value less than 0.05 that was set as the significance threshold.

An equal amount of protein from treatment samples was denatured by heating for 10 min at 95 °C in the Laemmle sample buffer. The denatured samples were loaded onto a 10% bis-tris, 26-lane gel (BioRad) and run at 200 V for 1.2 h. The gels were transferred onto PVDF membranes using the iBlot dry transfer system (ThermoFisher) and were blocked using 5% Milk in tris-buffered saline with Tween-20 (TBST). Primary antibodies used were as follows: anti-APP (clone 22C11; Millipore, 1:1000), anti-actin (Sigma, 1:500 000), anti-tubulin (Sigma, 1:500 000). After TBST wash, goat anti-mouse (1:3000) secondary antibody conjugated to horseradish-peroxidase was applied for 1 h. After washing, chemiluminescence “Super Signal” reagent (Pierce) was used to visualize protein bands.