Quantity one image analysis program

We used the LC3B monoclonal antibody (Cell Signaling, Danvers, MA, USA; #3868) at 1:1000 dilution, SQSTM1/p62 monoclonal antibody (Cell Signaling, Danvers, MA, USA; #5114) at 1:1000 dilution, and a beta-Actin antibody (Sigma, St Louis, MO, USA; #A5441) at 1:5000 dilution for the quantitative Western blotting analysis as described by Xie et al. [44 (link)]. We analyzed the signal intensity using the Quantity One image analysis program (Bio-Rad, Hercules, CA, USA). We used beta-Actin to standardize the amounts of protein (e.g., calculating the ratio of the amount of LC3-II to the amount of beta-Actin) and to limit disparities in the quantity of protein loaded. We expressed the protein levels as a percentage when comparing them to those in the control condition.

The quantitative Western blot was utilized in the present studies. We used antibody AT8 (55 ​kDa, 1:1000; Invitrogen, Carlsbad, CA) to detect the amounts of Tau phosphorylated at serine 202 and threonine 205 (Tau-PS202/PT205) amino acid. IL-6 antibody (24 ​kDa, 1:1000; Cat. # ab6672, Abcam, Cambridge, MA) was used to recognize IL-6. Postsynaptic density (PSD)-95 antibody (95 ​kDa, 1:1000; Cell Signaling, Danvers, MA), synaptophysin antibody (38 ​kDa, 1:1000; Cell Signaling) and N-cadherin antibody (140 ​kDa, 1:1000; Cell Signaling) were used to detect the protein amounts of PSD-95, synaptophysin, and N-cadherin, respectively. A β-Actin antibody (42 ​kDa, 1:5000; Sigma) was used to detect β-Actin. The quantification of Western blot was accomplished as described in other studies (Dong et al., 2009 (link)). In brief, we analyzed the signal intensity via Quantity One image analysis program (Bio-Rad, Hercules, CA). Two steps were used to quantify the Western blots. At the first step, β-Actin was used to standardize protein amounts (e.g., calculating the ratio of PSD-95 as compared to β-Actin amount), limiting the differences in the protein amount loaded. At the second step, we expressed the protein amounts obtained from the treatment as a percentage to the control condition.