Anti flag m2 antibody f1804

DMEM (10100147) and fetal bovine serum (C11995500BT) were from Gibco. MG132 (HY-13259) and protease-inhibitor cocktail (HY-K0010) were from MCE. Lipofectamine™ 3000 Transfection Reagent (L3000015) was from Invitrogen. Cycloheximide (CHX) and N-ethylmaleimide (NEM) were from Sigma-Aldrich. The following antibodies were from Abcam: anti-E4B (ab126759), anti-ubiquitin (ab134953), anti-HA (ab182009). Anti-GAPDH antibody (60004-1-Ig) and anti-PYCR2 antibody (17146-1-AP) were from Proteintech. Anti-TRA2A antibody (GTX87998) was from GeneTex. Anti-MYC antibody (9B11) was from Cell Signaling Technology. Anti-FLAG (M2) antibody (F1804) and anti-FLAG(M2) affinity gel (A2220) were from Sigma-Aldrich. Goat anti-rabbit IgG-Alexa Fluor 790 antibody (111-655-144) and goat anti-mouse IgG- Alexa Fluor 790 antibody (115-005-072) were from Jackson ImmunoResearch. Protein G Agarose (16-266) was from Merckmilipore. Phenylmethanesulfonyl fluoride (PMSF, ST506) and RIPA Lysis Buffer (P0013C) were from Beyotime. Polyetherimide (AC04L091) was from Life-iLab.

Cell lines: HepG2, Hep3B, SK-Hep1, HEK293, HEK293T, HeLa, ZR75-1, MCF7, U87-MG, U2OS, LoVo, HCT116, A375, MeWo, H460, A549, and Caki-1 cell lines were purchased from American Type Culture Collection (ATCC, USA); Huh7 and KGN cell lines were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences China (Shanghai, China).
Plasmids: All of Flag-tagged plasmids are constructed in pCDNA3.0 vector. The lentivirus plasmids for overexpressing LOXL4 or its mutants are constructed in pCDH vector. Primers used to construct these above plasmids were listed in Supplementary material.
Antibodies: Antibodies of anti-HA (sc-7392), anti-LOXL4 (sc-66952), and anti-β-actin C4 (sc-47778) were purchased from Santa Cruz Biotechnology, Inc. (USA); anti-Flag M2 antibody (F1804) and anti-GST antibody (G7781) were purchased from Sigma-Aldrich (USA); anti-p-p53(S15) (#9284), anti-p-p53(S20) (#9287) and anti-acetyl-p53 (Lys382) (#2525) antibodies were purchased from cell signaling technology (USA); anti-p-p53 (S15) (# NB100–92601) was purchased form Novus (USA).

The following antibodies were used: anti-phospho-ERK (sc-7383) and anti-FAK (sc-557), obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho-Akt (Ser473) (4060s) and anti-Akt (9272s), obtained from Cell Signaling Technology (Danvers, MA, USA); anti-phospho-tyrosine (clone 4G10) (05-321) and anti-phospho-FAK (Tyr397) (abt135), obtained from Merck Millipore (Burlington, MA, USA); anti-ERK2 (bms-52068R), obtained from Bioss (Boston, MA, USA); anti-FLAG-M2 antibody (F1804), obtained from Sigma-Aldrich; anti-Ki-67 (ab15580), obtained from Abcam (Cambridge, UK); anti-GAPDH (abc2003), obtained from AbClone (Seoul, Korea); and horseradish peroxidase-conjugated goat anti-mouse IgG (K0211589) and rabbit IgG (K0211708), obtained from KOMA Biotech (Seoul, Korea). The generation of anti-PTK7 anti-serum was described previously [57 (link)].

Cell extracts for Western blotting were obtained by resuspending transfected cells in radioimmunoprecipitation assay (RIPA) buffer consisting of 50 mM Tris HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1%SDS, 2 mM EDTA, 1 mM DTT, and protease inhibitor (Sigma Aldrich), followed by centrifugation at full speed for 20 min and collection of the supernatants. Proteins were denatured by boiling in Laemmli buffer. After SDS-PAGE, the proteins on the gels were transferred onto nitrocellulose membranes, blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20, and stained with specific primary antibodies (anti-Flag antibody M2 F1804 (Sigma Aldrich), anti-beta-actin antibody A5441 (Sigma Aldrich) or rabbit anti-ALKBH5 antibody #ab174124 (Abcam)) to the indicated proteins followed by incubation with secondary antibody conjugated with horseradish peroxidase and detection with chemiluminescence reagents.

For a custom anti-rabbit Hsp70-K77-Ac antibody, rabbits were immunized with synthetic peptide-bearing acetylated human Hsp70 (Ab_72–81:RLIGRK-acFGDP) following standard procedures (Peptron and Abfrontier). This antibody was purified with antigen-specific affinity chromatography and blocked by non-acetylated synthetic peptide (Ab_72–81:RLIGRKFGDP) before use. Anti-Hsp70 antibody (C92F3A-5, ADI-SPA-810, 1:3,000) and anti-Hsc70 antibody (1B5, ADI-SPA-815, 1:3,000) were purchased from Enzo Lifescience. Anti-Hop antibody (D6E3, #5669, 1:1,000), anti-Hsp40 antibody (#4868, 1:3,000), anti-acetylated lysine (Lys-Ac) antibody (#9941, 1:1,000), anti-cytochrome c antibody (#11940, 1:3,000), anti-cox-4 antibody (#4844, 1:3,000), anti-caspase-9 antibody (#9508, 1:3,000), anti-cleaved caspase-3 antibody (#9661, 1:3,000) and anti-HDAC4 antibody (#7628, 1:3,000) were from Cell Signaling Technology. Anti-Hsp90 antibody (H-114, sc-7947, 1:3,000), anti-CHIP antibody (H-231, sc-66830, 1:3,000), anti-Myc antibody (9E10, sc-40, 1:3,000), anti-ARD1 antibody (FL-235, sc-33820, 1:3,000) and anti-GST antibody (B-14, sc-138, 1:3,000) were purchased from Santa Cruz. The anti-FLAG antibody (M2, F1804, 1:3,000) and anti-tubulin antibody (DM1A, T9016, 1:3,000) were from Sigma. Anti-GFP antibody (ab6556, 1:3,000) and anti-ubiquitin antibody (Z0458, 1:3,000) were from Abcam and Dako, respectively.

Anti-BNRF1 was kindly provided by Dr. Lieberman [59 (link)], and anti-BZLF1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies against BMRF1, BALF4 (glycoprotein B), and LMP1 were described previously [56 (link)]. Anti-STAT1 (#9172), anti-phospho-STAT1 (Tyr701) (#9171), and anti-GAPDH (#5174) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-STAT2 (#693302) and anti-phospho-STAT2 (Tyr631) (#619851) antibodies were purchased from Biolegend. Anti-HA antibody (3F10) (#11867423001)and anti-Flag antibody (M2) (F1804) were purchased from Sigma-Aldrich.
Immunoblotting was performed as described previously [60 (link)]. Densitometry was performed using ImageJ.
For surface staining, cells were incubated with anti-glycoprotein B antibody before fixation. Then cells were stained with Alexa 647-anti-mouse IgG (A-21235; ThermoFisher Scientific, Waltham, USA) on ice for 30 min. Antibody-stained cells were fixed overnight with 4% paraformaldehyde at 4°C. Subsequently, cells were treated with 0.1% Triton-X100/PBS at room temperature for 10 min. Cells were then stained further with PE-anti-BZLF1 antibody (sc-53904 PE; Santa Cruz Biotechnology) on ice for 30 min. Cells were analyzed using a BD Fortessa X-20.

RNAiMAX (Invitrogen, United States) and jetPRIME^TM were obtained from Polyplus-transfection Biotechnology Company (France). 4, 6-Diamino-2-pheny- lindole (DAPI), anti-poly (ADP-ribose) polymeras [poly(ADP-ribose)polymerasepoly(ADP-ribose)polymerasePARP] and MitoTrack Green were purchased from Beyotime Biotechnology (Nanjing, China). Endotoxin Free Plasmid Preparation Kits and EASY spin plus RNA extraction kit were purchased from Aidlab (China). pCMV-Myc, pRK5-FLAG, pDsRed-monomer-N1 and pEGFP-N1 plasmid vectors were obtained from Clontech. Anti-GAPDH (CW0100) antibody was obtained from Kangwei Biological Company (Beijing, China). Anti-FLAG M2 (F1804) antibody and Rabbit anti-ORAOV1 polyclonal antibodies (SAB4300898) were purchased from Sigma (United States). Anti-c-Myc (sc-40), anti-GFP (sc-9996) and anti- β-actin (sc-1616-R) antibodies were obtained from Santa Cruz Biotechnology (United States). Anti-IBDV VP2 McAb (Clone ID: EU0205, which specifically recognizes 394 to 410aa of VP2) was purchased from CAEU Company (Beijing, China). Anti-Caspase-3 (9610) was purchased from Cell Signaling Technology. Caspase-3, Caspase-8 and Caspase-9 colorimetric assay kits were obtained from BioVision (United States), and PE Annexin-V apoptosis detection kit was purchased from BD Pharmingen (United States).