Cofilin d3f9

Antibodies to APP (6E10, Covance, Madison, WI, USA), cofilin (D3F9, Cell Signaling, Danvers, MA, USA), phospho-cofilin (77G2, Cell Signaling), actin (AC-74, Sigma Aldrich, St. Louis, MO, USA), tubulin (TU-02, Santa Cruz Biotechnology, Dallas, TX, USA), Aβ (D54D2, Cell Signaling), GFAP (Invitrogen), synapsin I (Invitrogen), PSD95 (Abcam, Cambridge, MA, USA), Drebrin (Abcam), microtubule-associated protein 2 (Millipore, Billerica, MA, USA), HRP-linked secondary antibodies (Jackson Immunochemicals, West Grove, PA, USA), and fluorescently labeled secondary antibodies (Invitrogen) were obtained from the indicated sources. The mouse monoclonal anti-RanBP9 antibody was a generous gift from Professor Elizabetta Bianchi (Pasteur Institute, France). Synthetic Aβ1-42 peptide was purchased from American Peptide (Sunnyvale, CA, USA). Aβ1-42 oligomers were prepared as previously characterized.^{21 (link)} Briefly, Aβ1-42 powder was dissolved in hexafluoro-2-propanol (HFIP) at 1 mM for 30 min at room temperature, aliquoted to eppendorf tubes, allowed to evaporate overnight in fume hood, and subjected to speed vacuum for 1 h to remove traces of HFIP or moisture. To prepare Aβ oligomers, Aβ1-42 film was then dissolved in dimethyl sulfoxide (5 mM), and F-12 cell culture medium (without phenol) was added to a final concentration of 100 μM Aβ1-42 and incubated at 4 °C for 24 h.

Western blotting of isolated B cells was performed with a LI-COR Odyssey imaging system as previously described (16 (link)). Primary antibodies for LCK (#2752, 1:1,000) and Cofilin (D3F9, 1:2,000) (both from Cell signaling) were used for detection.

For Western Blot immunostaining procedure, the membranes were incubated with Akt, phospho-Akt (Ser473) (D9E), phospho-S6 (Ser235/236) (D57.2.E), phospho-4E-BP1 (Thr37/46) (236B4), phospho-mTOR (Ser248) (D9C2), β-Actin (13E5), Cofilin (D3F9) (HRP conjugate), GAPDH (D16H11) (HRP conjugate), α-Tubulin (11H10) (HRP conjugate), phospho-Rb (Ser807/811), phospho-Rb (Ser795), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E), phospho-MEK1/2 (Ser217/221) (all purchased from Cell Signaling, Beverly, MA, USA), anti-c-Myc (phospho S62), anti–Glucose Transporter GLUT1, and anti-MCT4 (both from abcam, Cambridge, UK). The blots were incubated with the HRP-conjugated secondary antibodies goat anti-rabbit IgG or goat anti-mouse IgG (both from Santa Cruz Biotechnology, Dallas, TX, USA).