Anti flag tag

The antibodies used for immunoblotting, immunoprecipitation, immunochemistry, and immunofluorescence in this study were as follows: anti-USP39, 1:1000 (IB; Abcam, Waltham, MA, USA, ab131244); 1:100 (IHC; Abcam, Waltham, MA, USA, ab131244); (RIP, Proteintech, Rosemont, IL, USA, 23865-1-AP); anti-Flag-Tag, 1:1000 (IB; #80010-1-RR, Proteintech, Rosemont, IL, USA); anti-His-Tag, 1:1000 (IB; Cell Signaling Technology, #12698); anti-PARP, 1:1000 (IB; Cell Signaling Technology, Danvers, MA, USA, #9542); anti-Caspase-3, 1:1000 (IB; Cell Signaling Technology, Danvers, MA, USA, #9662); and anti- $\beta$ -actin, 1:5000 (IB: Sigma, Louis, MO, USA, A5316).

Total protein separation and western blotting were performed as described previously [16 (link)]. Western blot analysis was performed using anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9661), anti-p-AMPK (Thr172) (#50081), anti-p-ERK1/2 (#4370), and anti-HA-Tag (#3724) antibodies purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3 (14600–1-AP), anti-P62/SQSTM1 (18420–1-AP), anti-phospho-Akt (Ser-473) (66444–1-Ig), anti-Akt (10176–2-AP), anti-mTOR (20657–1-AP), anti-P38 MAPK (14064–1-AP), anti-JNK (51151–1-AP), anti-ERK1/2 (16443–1-AP), anti-AMPK (10929–2-AP), anti-Nrf2 (16396–1-AP), anti-Keap1 (10503–2-AP), anti-FLAG-tag (66008–2-AP), anti-MYC-tag (60003–2-AP), and anti-β-actin (66009–1-Ig) antibodies were obtained from Proteintech Group Co., Ltd. (Wuhan, China). An anti-phospho-mTOR (Ser-2448) (ab109268) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-p-p38 (sc-7973), anti-p-JNK (sc-6254), and anti-Ub (sc-8017) antibodies were obtained from Santa Cruz Biotechnology., Inc. (Santa Cruz, CA, USA).

DC_AC50, a CCS inhibitor, was provided by the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences. U0126-EtOH (catalog number: S1102) was purchased from Selleck. Antibody against phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000 times dilution) (catalog number: 4370S), p44/42 MAPK (Erk1/2) (1:1000 times dilution) (catalog number: 4695S), phpspho-MEK1/2 (Ser217/221) (1:1000 times dilution) (catalog number: 9154S), MEK1/2 (1:1000 times dilution) (catalog number: 8727S), β-actin (1:1000 times dilution) (catalog number: 8457S), mouse IgG (1:3000 times dilution) (catalog number: 7076S), and rabbit IgG (1:3000 times dilution) (catalog number: 7074S) were from cell signaling technology. Anti-Superoxide Dismutase 4 (1:500 times dilution) (catalog number: ab167170) was from Abcam. Anti-Flag tag (1:1000 times dilution) (catalog number: 66008) was from proteintech. CCS shRNA was purchased from Open Biosystems, Huntsville, AL. The sequence of targeted CCS shRNA was as follows: 5′-CCGGCTGATTATTGATGAGGGAGAACTCGAGTTCTCCCTCATCAATA ATCAGTTTTTG-3′. Lipofectamine RNA iMAX was purchased from Invitrogen. The sequences of targeted CCS siRNA were as follows: sense: 5′-GUCUUGGUACACACCACUCUA-3′; Antisense: 5′-UAGAGUGGUGUGUACCAAGAC-3′.

The cells were lysed with cold RIPA Lysis buffer (Gibco, USA) and centrifuged for 10 min at 10,000 rpm. The protein concentration was measured with a BCA Protein Assay kit (Thermo Scientific Pierce, Rockford, IL, USA). Total proteins were separated by 12% SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% (w/v) non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C and then incubated with primary antibodies, including anti-p53 (Abcam, USA), anti-MDM2 (Bioworld, USA), anti-p21 (Bioworld, USA), anti-Mad2L1 (Bioworld, USA), anti-CDC20 (Bioworld, USA), anti-BUB1 (Proteintech, USA), anti-Flag-tag (Proteintech, USA), and anti-β-ACTIN (Abcam, USA), overnight. Membranes were then washed with TBST three times and were finally incubated with the horseradish peroxidase labeled anti-mouse (or anti-rabbit) IgG secondary antibody (dilution ratio was 1:5,000) for 40 min. Densitometric analysis was performed using Image Pro-Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

Total proteins were extracted from cultured cells by RIPA buffer. The results showed that the BCA detection method (Beyotime, Nantong, Jiangsu, China) quantitatively detected all proteins. The protein samples separated by PAGE (polyacrylamide gel electrophoresis) were transferred to the PVDF membranes (EMD Millipore, Billerica, Massachusetts, USA). The PVDF membranes were blocked in 5% skim milk for 1 h, then incubated with specific antibodies overnight at 4 °C, and finally incubated with the secondary antibodies for 2 h at room temperature. The membranes were visualized using enhanced chemiluminescence solution (Pierce Biotechnology, Inc. USA). The experimental results were analyzed by ImageJ analysis software. The following antibodies were recorded: anti-GAPDH, anti-FLAG-tag, anti-HIF1α, anti-YTHDF2 (Proteintech, Wuhan, Hubei, China), anti-AKT, anti-Phospho-AKT (Ser473), anti-ERK1/2, anti-Phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technologies, Danvers, MA, USA), anti-Phospho-mTOR (Ser2448), anti-mTOR, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail1, anti-METTL3, anti-METTL14.

L. lactis cultures (20 μL) in the stationary growth phase were added to 500 μL of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and centrifuged at 5000× g for 5 min at 4 °C. The pellets were resuspended in 500 μL of TBS containing rabbit polyclonal anti-FLAG-tag (1:500; Proteintech) or anti-CNL (1:1000) for the detection of Stx1B and CNL, respectively [43 (link)]. After 2 h of incubation at room temperature (RT) with constant shaking at 100 rpm, the cells were washed three times with 200 μL of TBS with 0.1% Tween-20 (0.1% TBST) and resuspended in 500 μL TBS containing an Alexa Fluor 488-labelled anti-rabbit antibody (1:2000; Cell Signaling Technology). After 2 h of incubation at RT with constant shaking at 100 rpm, the cells were washed three times with 200 μL of 0.1% TBST and finally resuspended in 500 μL TBS. The samples were analyzed using a flow cytometer (FACS Calibur; Becton Dickinson, Franklin Lakes, NJ, USA) with excitation/emission at 488/530 nm in the FL1 channel. The geometric mean fluorescence intensity of at least 20,000 L. lactis cells in the appropriate gate was measured.

The following antibodies were used in the study: anti-Fbw7 (for IHC) (H00055294-M02, Abnova, Taipei City, Taiwan), anti-Fbw7 (for western blots) (ab109617, Abcam, Cambridge, UK), anti-Stat3, 12640; anti-phospho-Stat3^Tyr705, 4113; anti-Ubiquitin, 3936 (Cell Signaling Technology, Beverly, MA, USA), anti-Myc tag, 16286–1-AP; anti-Flag tag, 66008–2-Ig; and anti-β-actin, 60008–2-Ig; anti-Myc, 10828–1-AP; anti-Notch, 10062–2-AP; anti-Jun, 10024–2-AP; anti-DEK, 16448–1-AP; anti-Mcl1, 16225–1-AP(Proteintech, Rosemont, IL, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Amresco (Dallas, TX, USA).