Pser19 mlc2

Cells were seeded on top of a collagen I matrix and immunostained as described^{12 (link)}. Cells were fixed with 4% formaldehyde, permeabilised with 0.3% Triton X-100, blocked with 5% bovine serum albumin (BSA), and stained with primary antibody pSer19-MLC2 (1:200, #3671, Cell Signalling), which was detected with secondary Alexa Fluor 488 or 647 antibodies (Life Technologies). F-actin was stained using Alexa Fluor 546-phalloidin (Life Technologies) and nuclei with Hoechst 33258 (Life Technologies). Antibodies were diluted in 5% BSA-PBS. Imaging was carried out on Zeiss LSM 510 Meta confocal microscope with Plan-Apochromat 40x/1.2 NA (water) objective lenses and Zeiss LSM 710 confocal microscope with Plan-Apochromat 40x/1.3 Oil or a Plan-Apochromat 63x/1.4 NA (oil) objective lenses (Carl Zeiss) and Zen software. Images were analysed using ImageJ. p-MLC2 fluorescence signal was quantified calculating the pixel intensity in single cells relative to the cell area.

Antibodies and concentrations used: pSer19-MLC2 (#3671; 1:200 immunofluorescence; 1:50 immunohistochemistry), DDR1 (#5583; 1:1000 immunoblot, 1:200 immunohistochemistry), DRP1 (#8570; 1:1000 immunoblot), MFF (#86668; 1:1000 immunoblot), MFN1 (#14739; 1:1000 immunoblot), pThr172-AMPK (#2535; 1:1000 immunoblot; 1:150 immunohistochemistry), AMPK (#2532; 1:1000 immunoblot), MYPT (#8574; 1:1000 immunoblot), pSer79-Acetyl-CoA Carboxylase (#3661; 1:1000 immunoblot), Acetyl-CoA Carboxylase (#3662; 1:1000 immunoblot), from Cell Signaling Technology; pSer172/Ser146-MFF (AF2365, 1:1000 immunoblot (Ser172 in MFF human isoform 1 corresponds to Ser146 in MFF human isoforms 2, 4 and 5)), pSer472-MYPT1 (AF3779; 1:1000 immunoblot), from Affinity Biosciences; GAPDH (MAB374; 1:10000 immunoblot) from Merck-Millipore; Tubulin (T6199; 1:10000 immunoblot) from Sigma-Aldrich; Integrin β1 (ab24693; 1:100 immunofluorescence), MFN2 (ab56889; 1:1000 immunoblot, 1:500 immunohistochemistry), CD44 (ab157107; 1:700 immunohistochemistry) from Abcam; OPA1 (612606; 1:1000 immunoblot, 1:500 immunohistochemistry) from BD Transduction Laboratories; Tom20 (sc-17764; 1:800 immunofluorescence) from Santa Cruz Biotechnology.

Antibodies and concentrations used: pThr18/Ser19-MLC2 (#3674; 1:750, immunoblot), pSer19-MLC2 (#3671; 1:50, immunohistochemistry; 1:200, immunofluorescence), MLC2 (#3672; 1:750), pT202/Y204-p44/42 (ERK1/2) (#4370; 1:1,000), pY705-STAT3 (#9145; 1:750), PD-L1 (clone E1L3N, #13684, 1:200) from Cell Signaling Technology; STAT3 (sc-482; 1:500), ERK2 (sc-154; 1:1,000), MCL-1 (sc-819; 1:1,000), GFP (sc-8334; 1:1,000) from Santa Cruz Biotechnology; GAPDH (MAB374; 1:10,000) from Millipore; P-H2A.X (S139) (ab2893;1:1000), CD206 (ab64693; 1:1,000), CD3 (anti-mouse, ab134096; 1:500), CD4 (anti-mouse, clone I3T4, ab183685; 1:300), FoxP3 (anti-human, clone 236A/E7, ab20034; 1:200) from Abcam; F4/80 (anti-mouse, clone BM8, MF48000, 1:1000), CD8a (anti-mouse, clone Ly2, 14-0808-82; 1:200), FoxP3 (anti-mouse, clone FJK-16s, 14-5773-82; 1:200) from Invitrogen; CD4 (anti-human, clone 11E9, NCL-L-CD4-368; 1:300) from Novocastra.