Ab192452

For immunofluorescence experiments [23 (link),26 (link)], cells were processed as previously described, with slight modifications [23 (link),26 (link)]. Briefly, THP-1 macrophages were incubated for 1 h and immediately fixed with pre-warmed 3.7% formaldehyde (15 min, room temperature). After permeabilization (0.2% Triton-X-100, 10 min), unspecific binding sites were blocked (2% Donkey Serum, 0.5% Bovine Serum Albumine, 1 h). Afterwards, cells were incubated for 2 h with anti-Caveolin-1 antibody (Abcam, ab192452, 1:500), anti-TLR4 antibody [76B357.1] (Abcam, ab22048, 1:200) and anti MIF antibody (Abcam, ab7207, 1:500). After 5 washing steps, species-specific fluorescence-labeled antibodies were used for localization (Donkey Anti-Goat IgG (H+L), Alexa Fluor^® 647 (705-605-003) and Donkey Anti-Mouse IgG (H+L) Alexa Fluor^® 488 (715-545-150) from Jackson ImmunoResearch Laboratories, Inc., Pennsylvania, US and Donkey anti-Rabbit IgG (H+L) polyclonal secondary antibody, Alexa Fluor^® 568 (A10042) from Thermo Fisher Scientific, Waltham, US).

InSolution Rapamycin [Calbiochem 553,211; 1–100 nM (Foster and Toschi 2009 (link))], bafilomycin A1 [bafilomycin, SML-1661; 10 nM (Redmann et al. 2016 (link))] and Nicotinamide [N0636; 0.1–5 mM Avalos et al. 2005 (link); Hwang and Song 2017 (link))], Yoda1 [REF: SML 1558; 5 µM (Davies et al. 2019 (link))], Filipin complex ready-made solution (REF: SAE0088) were purchased from Sigma-Aldrich (USA). The compounds were dissolved in DMSO or water according to the technical specification of the suppliers. Solvent controls were matched to represent the same solvent concentrations of the treatments which were obtained by diluting stock solutions 1:1000. For the immunofluorescence studies, the following antibodies were used: anti-acetylated Lysine Monoclonal Antibody (1C6) (MA1-2021, Invitrogen), anti-Caveolin-1 (ab192452, Abcam, dil 1:500), anti-Integrin beta 1 (ab30394, Abcam, dil 1:500), anti-PMP70 (PA1-650, Invitrogen dil 1:500), anti-CLUH (PA5-65,101, Invitrogen, dil 1:500), anti-KLF2 (ab203591, Abcam, dil 1:600), anti-TOM20 (sc-17764, Santa Cruz Biotechnology, dil 1:500), Alexa Fluor® 647 donkey anti-mouse IgG (H + L) (A31571, Invitrogen, dil. 1:1000), Alexa Fluor® 647 donkey anti-goat IgG (H + L) (A21447, Invitrogen, dil. 1:1000), Alexa Fluor® 568 donkey anti-rabbit IgG (H + L) (A10042, Invitrogen, dil. 1:1000), Oregon Green® 488 Phalloidin (O7466, Invitrogen, dil. 1:1000).

Protein localization was performed in tissue sections of gracilis muscle from control rats. Briefly, gracilis muscles were fixed with 2% paraformaldehyde and embedded in paraffin for tissue sectioning. Five-micron-thick sections were blocked with 5% normal donkey serum and incubated overnight with mouse anti-BK (1:200, Abcam [ab99046]), rabbit anti-TRPV4 (1:100, Abcam [ab39260]), or goat anti-caveolin-1 (Cav-1; 1:200, Abcam [ab192452]). All sections were treated with mouse or goat anti-alpha actin (1:300). Tissue sections were then incubated with appropriate secondary antibodies for 20 min at RT. 633 hydrazide (1 μm) was used to stain the internal elastic lamina. Samples were mounted with anti-fade mounting media and images were acquired using a confocal microscope (TCS SP5; Leica Microsystems).