Alexa 647 conjugated goat anti mouse igg

Anti-β-actin (ab179467), anti-NMDAR-2B (ab65783), anti-PSD-95 (ab18258), anti-SYP (ab32127), anti-SQSTM1/p62 (ab56416), anti-SOD1 (ab179843) and anti-SIRT1 (ab32441) were acquired from Abcam (Cambridge, UK). Anti-LC3A/B (#4108), anti-PI3K (#4255), anti-Akt (#4691), anti-phospho-Akt (#4060), anti-mTOR (#2983) and anti-phospho-mTOR (5563) were acquired from Cell Signaling Technology (Danvers, MA, United States). Anti-Beclin-1(sc-48341) was acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States).
Anti-β-amyloid (# SIG-39142) was acquired from BioLegend (San Diego, CA, United States).
The secondary antibodies of Alexa 488-conjugated goat anti-rabbit IgG, Alexa 647-conjugated goat anti-rabbit IgG and Alexa 647-conjugated goat anti-mouse IgG were acquired from Invitrogen (San Diego, CA, United States).

Immunolabeling of control siRNA and talin-1 siRNA-transfected H1299-P/L cells for talin was performed with a liquid-handling robot (Model FREEDOM EVO, Tecan, Switzerland) to minimize the variance between experimental repeats. Briefly, the cells were fixed in 2% paraformaldehyde for 10 min at RT, washed once with PBS, and permeabilized with 0.1% Triton X-100 for 5 min. Following 4Â washing, cells were blocked by incubation with 1% BSA/PBS. Then cells were incubated with an anti-talin antibody (1 : 100, clone 8d4, Sigma-Aldrich) for 30 min, washed 4Â with PBS, and incubated with Alexa 647-conjugated goat anti-mouse IgG (1 : 300, Invitrogen, USA) for 30 min at RT. The cells were washed with PBS 4Â before the plate was re-imaged at the microscope. Notably, immunofluorescent labeling of talin was optimized to retain the total cytoplasmic talin pool, as opposed to the canonical optimization for adhesion labeling. Such adhesion-specific labeling could also be achieved with the same antibody using a simultaneous fixation and permeabilization condition (Fig. S2A,ESI †). Specificity of the talin antibody was confirmed by comparison with secondary antibody (Alexa 647-conjugated goat anti-mouse IgG) labeling only (Fig. S2B and C, ESI †).