Mouse anti actin

Secretion assay was performed as described by Djamei et al. (2011). In brief, after filament induction, the cell‐free supernatant was precipitated with trichloroacetic acid (TCA)/Sodium deoxycholate (DOC) and a Western blot was performed on the precipitate. Ustilago maydis strain AB33 P_otefapB73‐HA was generated by insertion of plasmid p123‐P_otef‐ApB73‐HA into the ip locus of AB33 (Brachmann et al., 2001). AB33 cell extracts and supernatants were used to ensure specificity of the antibodies. Actin detection served as a lysis control and bands could only be observed for whole cell extracts. For Western blot analysis, mouse anti‐haemagglutinin (anti‐HA) (Sigma‐Aldrich, St. Louis, MO, USA) and mouse anti‐actin (Invitrogen, Waltham, MA, USA) antibodies were used. Assays were carried out at least three times.

Protein was collected from the cells lysed in RIPA buffer with the protease inhibitor (Roche). Protein extracts were passed through a 25g needle to break up DNA and subsequently quantified using the BCA assay. Ten microgram of total protein was loaded per lane onto a 12% SDS-PAGE gel (Bio-Rad), which was run at 100 V for 2 h. The proteins were transferred to a 0.2-μm PVDF membrane (Bio-Rad) using Trans-Blot Turbo (Bio-Rad) at 1.3 A for 7 min. Blots were incubated in primary antibodies (sheep anti-BPNT2, 1:1000, Invitrogen, #PA5-47893; mouse anti-Actin, 1:1000, Invitrogen, #MA1-744) overnight at 4 °C, washed 3 times in 0.1% TBS-Tween, then in secondary antibodies (AlexaFluor680 anti-sheep 1:20,000; AlexaFluor800 anti-mouse 1:20,000) for 2 h at room temperature, and washed 3 times in 0.1% TBS-T. The blots were imaged on LI-COR Odyssey.

To assess expression of endogenous or transduced proteins, cell lysates containing 30–40 μg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: mouse anti-TMPRSS2 (Santa Cruz, #515727, 1:1000), mouse anti-Cathepsin-L (Santa Cruz, #32320, 1:1000), goat anti-ACE2 (R&D systems, #AF933, 1;1000), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-MAVS (Thermo Fisher, #PA5-17256, 1:1000), mouse anti-RIG-I (AdipoGen, #20B-0009, 1:1000), rabbit anti-MDA-5 (Proteintech, #21775-1-AP, 1:1000), rabbit anti-UNC93B1 (Invitrogen, #PA5-83437, 1:1000), rabbit anti-IRF1 (Cell Signaling, #8478S, 1:1000). Specific staining was visualized with secondary antibodies, goat anti-mouse-IgG-DyLight 680 (Thermo Scientific, #35518, 1:20000), goat anti-rabbit-IgG-DyLight 800 (Thermo Scientific, #SA5-35571, 1:20000), or a donkey anti-goat-IgG-IR-Dye 800 (Licor, #926–32214, 1:20000). As loading controls, actin or tubulin expression was probed using a rabbit anti-actin (Sigma-Aldrich, A2066, 1:5000), mouse anti-actin (Invitrogen, #AM4302, 1:5000), or rabbit anti-tubulin (Cell Signaling, #3873, 1:5000). Membranes were scanned with an Odessy scanner (Li-Cor).

Protein extracts were prepared from heads or dissected brains. Cell homogenization was done in RIPA buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0, supplemented with protease inhibitor cocktail from Roche Life Science). After 2 hrs of lysis at room temperature, Laemmli buffer was added before to boil the samples during 5 min.
Samples were analyzed by SDS-PAGE. To separate alpha and beta-tubulins, we used a protocol described by Banerjee and collaborators^{63 (link)}. Separated proteins were electrophoretically transferred onto nitrocellulose membrane (Hybond C-Extra, Amersham Biosciences) prior to blotting. Primary and secondary antibodies were incubated in 5% milk in PTX (PBS, 0.1% TritonX100) and washes were done with PTX. Immunodetection was done with Clarity Western ECL kit (BIO-RAD). Chemiluminescence detection was acquired with ChemiDoc Touch Imaging System (BIO-RAD). The following primary antibodies were used: mouse GT335 (1/200, AdipoGen), mouse 1D5 (1/500, Synaptic System), mouse anti-acetylated-tubulin (1/2000, Sigma), mouse DM1A (1/4000, anti-alpha tubulin from Sigma), mouse E7 (1/1000, anti-beta tubulin from DSHB), mouse anti-actin (1/2000, ThermoScientific). Secondary antibody used was HRP-linked goat anti-mouse (1/10000, Jackson ImmunoResearch).

Cell vulnerability assays were performed as it follows. Low-passage normal (STHdh7Q) and mutant (STHdh109Q) Htt mouse striatal cells^{20 (link)} were cultured in 24-well plates and cell vulnerability assays performed as previously described using serum deprivation for cell death induction^{97 (link)}. Serum deprivation was performed by replacing the medium with 1% FBS medium and with drug or vehicle. Cell vulnerability was scored 24 hours later upon DAPI staining and by counting pyknotic versus normal nuclei in DAPI-positive cells. This involved three independent experiments with 100 scored cells each, and assays were performed blindly. For Western blotting, proteins were extracted as previously described^{97 (link)}, separated by SDS-PAGE, and analyzed by Western blotting using the following primary antibodies: mouse anti-HTT (4C8, Chemicon, 1:5,000) and mouse anti-actin (ThermoFisher, 1:5,000). The secondary antibody used was goat–anti-mouse IgG HRP-conjugated (Biorad, 1:10,000). Proteins were detected by using ECL + (ECL for actin) and evaluated by densitometry. At least three independent assays were performed. Signals were quantified using ImageJ.