Journal: bioRxiv

Article Title: Dynamic S-acylation controls CMG2 maturation, extracellular matrix regulation, and anthrax toxin entry

doi: 10.64898/2026.01.06.697925

Figure Lengend Snippet: a. Representative confocal images ofRPE1 cells, WT or knocked for APT2. Cells were incubated 1 hour at 4°C with 500 ng/ml Anthrax toxin Protective Antigen (PA) and 100ng/ml Lethal Factor LF; cells were washed and incubated for 2h30 at 37°C. Mek2 was stained by immunofluorescence, and nuclei were stained with Hoechst. Scale bar = 10μm. b. Quantification of a . Cells were segmented using Cellpose3 and Mek2 mean staining intensity was measured for each cell. Groupped were compared using a Kruskal-Wallis test, ***: p-value < 0.0001. c . RPE1 cells were pre-incubated 4 hours at 37°C with 10µg/ml ML348 or ML349. Cells were incubated 1 hour at 4°C with 500µg/ml PA and 50ng/ml LF and chased different indicated times at 37°C. 40 µg of protein samples were submitted to SDS-PAGE, and analyzed by immunoblotting with rabbit anti-human CMG2, rabbit anti-PA or rabbit anti-MEK2 N-terminal. The amount of PA pore (SDS-resistant) ( d ) and cleaved MEK2N-terminal ( e ) were quantified, and set to 1 after 1 hour at 4°C before chase, and different time of chase were expressed relative to this (n=4, error bars represent standard deviation). f. RPE1 cells were silenced for 3 days with human APT2 RNAi or control RNAi. Cells were changed 24h prior experiment in serum free medium, incubated with 1ug/ml Collagen6 indicated hours. 40 °g of protein samples were submitted to SDS-PAGE, and analyzed by immunoblotting with rabbit anti-collagen VI, anti-CMG2, and anti-IgG rabbit. g. Cartoon of the S-acylation and deacylation events during the life cycle of CMG2.

Article Snippet: The other antibodies were commercially available: goat anti-human CMG2 (R&D, AF2940, RRID: AB_2056740); rabbit anti-human CMG2 (Proteintech, RRID: AB_2056741); goat anti-human TEM8 (R&D, ref: AF3886, RRID: AB_2056726); rabbit anti-ARF6 (LSBio, ref: LS-C81881,RRID: AB_2058487); mouse anti-TRAP alpha (Santa Cruz, RRID: AB_1130631); anti-V5 (Invitrogen, 46-0705, RRID: AB_2556564); anti-GFP (Takara Bio, RIDD: AB_11153295); mouse anti-Myc (SIGMA, 9E10 M4439, RRID: AB_439694), anti-human ZDHHC3 (Abcam, ab31837, RRID: AB_742236); anti-GM130 (BD biosciences, 610823, RRID:AB_398142); mouse anti-GAPDH (Acris Antibodies, 4A1-MA0100, RRID_AB1874646); anti-actin (Merck Millipore, RRID: AB_2223041); anti-Talin (Sigma, RRID: AB_477572); anti-Vinculin (Sigma, RRID: AB_477629); anti-phospho tyrosine (Millipore, RRID: AB_916371); anti-RhoA (Cell signalling, 67B9, RIDD: AB_10693922); anti-SRC (Abcam, ref: 16885, RRID: AB_443522); anti-Anthrax Protective Antigen (List Biological Laboratories, ref: 771B); anti-MEK2 (Santa Cruz, ref:12578, RRID: AB_648958), anti-collagen VI (Fitzgerald laboratories, ref: 70R-CR009X, RRID: AB_1283876).

Techniques: Incubation, Staining, Immunofluorescence, SDS Page, Western Blot, Standard Deviation, Control

Journal: Disease Models & Mechanisms

Article Title: Col6a1 knock-in mice provide a promising pre-clinical model for collagen VI-related dystrophies

doi: 10.1242/dmm.052460

Figure Lengend Snippet: Quantitative analysis of immunoblotting for collagen alpha 1 (VI). Bars graphs showing the relative abundance of collagen alpha 1 (VI) in triceps, diaphragm, gastrocnemius and tibialis anterior muscles of wild-type, heterozygous and homozygous mice at different ages ( n =3-6 mice per genotype per stage). Col6a1 was detected with a polyclonal antibody against collagen type VI (70R-CR009x). Data were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. Data are the mean±s.e.m. * P <0.05, ** P <0.011, *** P <0.001, **** P <0.0001. WT, wild-type (Col6a1 +/+ ); HET, heterozygous (Col6a1 G292R/+ ); HOM, homozygous (Col6a1 G292R/ G292R ).

Article Snippet: Blotted membranes were blocked for 1 h in 5% milk or BSA in Tris-buffered saline [10 mM Tris, 150 mM NaCl (pH 8.0)] plus 0.1% Tween and incubated overnight at 4°C with rabbit polyclonal anti-collagen VI (Fitzgerald, #70R-CR009X) or rabbit polyclonal anti-desmin (Abcam, #ab8592) antibodies.

Techniques: Western Blot, Muscles, Comparison