Rabbit anti myc antibody

Flow cytometry data was acquired on a FACSVerse using FACSuite software or FACSCanto, or LSR II Fortessa with FACS DIVA software (BD Biosciences). Data was analysed using Flowjo software version 9.9.6 (Treestar).
For cell surface staining antibodies conjugated to BV421, BV510, FITC, PE, PerCPCy5.5, PECy7, APC, and APCeF780 were obtained from BD Biosciences, eBioscience or Biolegend. Fc receptors were blocked using Fc block (BD Biosciences). Antibody clones were as follows: CD4 (RM4-5), CD8 (53–6.7), CD11b (M1/70), CD25 (7D4), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD69 (H1.2F), TCRbeta (H57-597), Thy1.2 (53–2.1), B220 (RA3-6B2), NK1.1 (PK136).
For Myc intracellular staining, cells were fixed and permeabilised overnight in PBS 1% FBS 0.5% PFA 0.2% Tween-20. Fix/perm was washed off and cells were stained with 1:200 rabbit anti-Myc antibody (Cell Signalling Technologies, clone D84C12, cat#5605S) for 1 hr at room temperature followed by 1:1000 anti-rabbit IgG (H+L) F(ab’)₂ AlexFluor647 secondary antibody (Cell Signalling Technologies, cat#4414S) for 1 hr at room temperature.
For IFNγ and Granzyme B intracellular staining cells were fixed and permeabilised using eBioscience Intracellular Fixation and Permeabilisation kit (eBioscience) as per manufacturer instructions. Cells were stained with anti-IFNγ (XMG1.2) and anti-Granzyme B (NGZB) at 1:100 and 1:200 respectively.

Rabbit anti-ACAT2 (1:1000) (ab131215) and rabbit anti-P21 (1:1000) (ab109520) were purchased from Abcam. Rabbit anti-SETD7 antibody(1:1000) (24840-1-AP), rabbit anti-P16-INK4A(1:1000) (10883-1-AP), rabbit anti-P27 (1:1000)(25614-1-AP), rabbit anti-Cyclin B1 (1:1000)(55004-1-AP), rabbit anti-Cyclin E1(1:1000)(11554-1-AP), mouse anti-Cyclin D1(1:1000)(60186-1-Ig), rabbit anti-MCM2(1:1000)(10513-1-AP), rabbit anti-Cortactin (1:400)(11381-1-AP) and rabbit anti-SMA (1:1000)(14395-1-AP), mouse anti-Vimentin(1:4000)(60330-1-Ig) were purchased from Proteintech. Rabbit anti-Snail2 (1:1000) (121235) was purchased from Brickell Biotech, Inc. Antibodies against YAP1(1:1000) (A1002), TAZ (1:1000) (A23034), TEAD1(1:1000) (A5218) were purchased from AB clonal. Rabbit anti-vinculin (1:1000) (E1E9V), rabbit anti-flag antibody (1:1000) (2272S), rabbit anti-myc antibody (1:1000) (14793S), rabbit anti-p53 (7F5) (1:1000)(2527S), rabbit anti-E-Cadherin (24E10) (1:1000)(3195S), rabbit anti-N-Cadherin (D4R1H) XP®(1:1000)(13116S) and mouse anti-ubiquitin (1:1000) (#3936) were purchased from Cell Signalling Technology.
MG132 (HY-13259), Cycloheximide (CHX) (HY-12320) and Cell Counting Kit-8 (CCK-8, HY-K0301) were purchased from MedChemExpress (Shanghai, NJ, USA).

Protein stability assays were performed according to established methods54 (link). In brief, HEK293T cells were transfected using Xtreme-Gene (Roche 6366546001) following the manufacturer's instructions. After transfection, cells were incubated for 48 h in DMEM media supplemented with 10% FBS, then were treated with 100 μg/ml cycloheximide (2112, Cell Signaling) in DMEM media with 10% FBS. At the indicated times. 100 μl of 2X SDS-PAGE sample buffer was added and the cells were scraped from the wells, boiled for 5 min, then cell lysates were stored at −80 °C. After all lysates were collected, each sample was loaded onto a 10% SDS-PAGE gel and then analysed by western blotting with rabbit anti-MYC antibody (5605, Cell Signaling, 1:1,000) to monitor MYC protein level. Protein expression was quantified from the western blot using GelQuant software. For analysis, MYC levels were normalized to tubulin protein levels (mouse, T-5326, Sigma, 1:2,000). Assays were performed three times.

All antibodies were diluted in PBS with 1% skimmed milk. Human monoclonal antibodies against SARS-CoV-2 S (S2 subunit) were kindly provided by Florian Klein, University of Cologne (Köln, Germany) (HbnC5t1p1_B10, HbnC5t1p1_E5). We stained cis-Golgi compartments with a monoclonal mouse anti-GM130 antibody (BD Bioscience, #610822, dilution 1:5) and ERGIC with a monoclonal mouse anti-LMAN1 antibody (Invitrogen, #PA1-074, dilution 1:50). Myc-tagged Rab1A and Rab1B proteins were stained with a rabbit anti-Myc antibody (Cell Signaling, #2272S, dilution 1:50 for immunofluorescence assays (IFAs) and 1:250 for Western blot). Endogenous tubulin was stained with a monoclonal antibody from Sigma-Aldrich (#T9026, dilution 1:500). Alexa Fluor-labeled secondary antibodies were purchased from Invitrogen (Anti-human-Alexa Fluor 488, #A-11013; anti-mouse-Alexa Fluor 568, #A-11004; anti-mouse-Alexa Fluor 594, #A-11032; anti-rabbit-Alexa Fluor 594, #A-11012; anti-rabbit-Alexa Fluor 647, #A27040, secondary antibodies were diluted 1:400). For Western blot analyses, following HRP-coupled secondary antibodies were used: Anti-human (Invitrogen, Waltham, MA, USA, #PA1-28587, dilution 1:3000), anti-rabbit (Dianova, Geneva, Switzerland, #711-036-152, dilution 1:40,000) and anti-mouse (Dako, Glostrup, Denmark, #P044701-2 dilution 1:40,000).

We used the phumanTUBB1‐tagged Myc vector described by Kunishima et al (2009). Mutant P160L‐TUBB1 was generated using a PCR‐based site‐directed mutagenesis method as described previously, using the Stratagene QuikchangeVR Kit (Agilent Technologies; Carré et al, 2007). Nthy (Nthy‐ori 3.1; given by Corinne Dupuy) immortalized human thyroid‐cell lines were cultured as previously described and used from passage 12 (Lemoine et al, 1989). The Nthy cells were plated at 0.4 × 10⁵/well on poly‐l‐lysine‐coated slides in 12‐well plates 24 h before transfection then transfected with 500 ng of vectors containing wild‐type or P160L mutant TUBB1 using XtremeGENE‐HP‐DNA, as recommended by the manufacturer (Roche Applied Science, Penzberg, Germany). After 24 h, cells were used for immunofluorescence as already described (Bourg et al, 2015). The cells were washed with pre‐warmed PHEM buffer; fixed with 4% PFA, 0.2% glutaraldehyde and 0.5% Triton; and permeabilized with PBS‐Triton 0.1%. Immunostaining was performed with rabbit anti‐Myc antibody (# 2272, 1:500, Cell Signaling Technology) and mouse anti‐α‐tubulin (DM1A, # T9026, 1:1,000, Sigma‐Aldrich, Saint‐Louis, MI, USA) then with Alexa Fluor 647 goat anti‐rabbit and Alexa Fluor 555 goat anti‐mouse antibodies (1:400, Thermo Fisher Scientific).