2.10. Western Blot Analysis of p-LRRK2, LRRK2, IL-6, IL-10, IL-1β, IL-13, TNF-α, GYS, and p-GSK3-α/β

Western blot analysis allowed to evaluate the expression of p-LRRK2, LRRK2 proteins, and those involved in inflammatory processes such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, IL-13, and IL-10. Western blot analysis was performed as previously described [13]. Membranes were incubated at 4 °C overnight with each of the previously mentioned primary antibodies diluted in milk [17], PBS, and 0.1% Tween-20 (PMT): antiphosphorylated LRRK2 at Serine 935 (p-Ser935) (1:1000, Abcam ab133450); anti-LRRK2 (1:1000, Abcam ab133474); anti-IL-1β (1:500; Santa Cruz Biotechnology sc-32294), anti-TNF-α (1:500; Santa Cruz Biotechnology sc-52746), enzyme glycogen synthase (GYS) (1:500; Santa Cruz Biotechnology, sc 81173), glycogen synthase kinase 3 (GSK-3-α/β) (1:500; Santa Cruz Biotechnology, sc 7291), and phosphorylated GSK-3-α/β (p-GSK-3-α/β) (1:500; Santa Cruz Biotechnology, sc 81496). Finally, membranes were incubated for 1 h at room temperature with a secondary antibody and bands were obtained using a chemiluminescence detection system (ECL) according to the manufacturer’s instructions (Thermo, Waltham, MA, USA). Protein expression was quantified by band densitometry and standardized to GAPDH (anti-GAPDH antibody 1:500; sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA) levels as an internal control. The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTMXRS + software.