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Y., Goodman C. 15). The EHD proteins regulate endocytosis of receptors and their recycling to the plasma membrane after internalization (16,C23). EHD proteins are characterized by an amino-terminal ATPase domain as well as a carboxyl-terminal EH domain; the EH domain is an EF hand-like structure that interacts with proteins containing an asparagine-proline-phenylalanine (NPF) motif (24,C27). Myoferlin harbors an NPF motif in its C2B domain, and this region was shown to mediate EHD2 binding (14). Reduction of EHD1 in human cells impairs transferrin recycling (28). Myoferlin-null myoblasts accumulate more labeled transferrin initially and are less efficient at recycling the transferrin receptor to the plasma membrane than control myoblasts (14). We have now characterized Fer1L5, the only other mammalian Benfotiamine ferlin to contain an NPF motif. We found that Fer1L5 is expressed in myoblasts undergoing fusion to myotubes and that Fer1L5 can bind both EHD1 and EHD2, two EHD family members that are also expressed in myoblasts. siRNA-mediated reduction of EHD1 and/or EHD2 expression leads to impaired myoblast fusion. Reduction of EHD2 protein levels inhibits normal transit of Fer1L5 through the secretory system to the plasma membrane. EHD proteins and ferlin proteins form discrete structures in myoblasts. From these data, we propose a model where multiple ferlin proteins interact with EHD proteins to mediate the cytoskeletal rearrangements necessary for proper membrane recycling and myoblast fusion. EXPERIMENTAL PROCEDURES Cell Culture C2C12 Hsh155 cells were obtained from ATCC (catalogue number CRL-1772). The cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 7.5% CO2. The cells were differentiated in DMEM supplemented with 2% horse serum and 1% penicillin/streptomycin in 7.5% CO2. All of the tissue culture media and sera were from Invitrogen. Immunoblotting and Immunostaining For immunoblot time course analysis, C2C12 cells were plated at equal densities on 10-cm tissue culture plates and harvested at specified time points. The cultures were lysed in 1 ml of lysis buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 1% Triton, 1% Halt protease inhibitor mixture (Pierce), and PMSF). The lysates were centrifuged at Benfotiamine 14,000 for 15 min at 4 C to remove cellular debris, and the protein concentration of the supernatant was determined using a Bio-Rad protein assay. Fifty g of protein was separated on a 4C20% acrylamide gel stained with GelCode Blue stain reagent (Pierce) or transferred to PVDF Immobilon-P membrane (Millipore, Billerica, MA). The membrane was immunoblotted with rabbit polyclonal anti-myoferlin at 1:3000 (MYOF3) (10), mouse monoclonal anti-dysferlin at 1:3000 (NCL-Hamlet; Novocastra Ltd.), and rabbit polyclonal anti-Fer1L5 antibody (ab1005) at 1:3000. Peptides for anti-Fer1L5 antibody production were selected using MacVector. The peptide EQKDQPRKEMEKTRSWQPWK (amino acids 1031C1050) was synthesized, coupled to keyhole limpet hemocyanin, and injected into rabbits (Bethyl Laboratories, Montgomery, TA) to generate anti-Fer1L5 ab1005. A second antibody, anti-Fer1L5 ab412 was generated against the peptide sequence RGGKKPPFRTSEEGTCIMDA (amino acids 438C457). The specificity of the antibodies was tested by blocking Benfotiamine the immunostaining from 2 g of antibody with 40 g of respective peptides. Other than this specificity test, ab412 was not used. Secondary antibodies, goat anti-rabbit and goat anti-mouse antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA), were used at a dilution of 1 1:5000. Blocking and antibody incubations were performed in StartingBlock T20 blocking buffer (catalogue number 37543; Pierce). ECL-Plus chemiluminescence (Amersham Biosciences) and Kodak Biomax MS film were used for detection. For immunostaining analysis, C2C12 cells had been plated at identical densities on NaOH-washed cup coverslips within 6-well plates. The cells had been set in 4% paraformaldehyde for 10 min. Blocking and antibody incubations had been performed in 1 PBS filled with 5% fetal bovine serum. Rabbit polyclonal anti-Fer1L5 was utilized at 1:100, rabbit polyclonal MYOF3 was utilized at 1:100, mouse monoclonal NCL-Hamlet was utilized at 1:200, goat polyclonal anti-EHD2 (ab23935, Abcam, Cambridge, MA) was utilized at 1:500, rabbit polyclonal anti-EHD1 (17) was utilized at 1:100, and rabbit polyclonal anti-EHD4 was utilized at 1:100. Donkey anti-rabbit conjugated to Alexa 488, donkey anti-goat conjugated to Alexa 594, goat anti-rabbit conjugated to Alexa 594, goat anti-mouse conjugated to Alexa 488, phalloidin conjugated to Alexa 488, and phalloidin conjugated to Alexa 633 (Molecular Probes) had been utilized at 1:2000. The coverslips had been installed using Vectashield with DAPI (Vector Laboratories, Burlingame, CA). The pictures were captured utilizing a Leica TCS SP2 AOBS Laser Benfotiamine beam Checking Confocal (Leica Microsystems Inc., Bannockburn, IL) or a Zeiss Axiophot microscope and Axiovision software program (Carl Zeiss, Maple Grove, MN). To quantify ferlin appearance in a.