Goat anti mouse igg horseradish peroxidase hrp

The total proteins were extracted from the mice and A549 cells by PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Inc., Seongnam, Korea). Protein was extract by PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Inc., Seongnam, Korea). An equal amount of total protein (20 μg) was resolved on 8–15% sodium dodecyl sulfate polyacrylamide gel and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membranes were blocked for 1 h in 2.5% skim milk solution and incubated overnight at 4 °C with specific antibodies. To detect target proteins, specific antibodies against BDNF (abcam, #ab108319), TrkB (abcam, #ab18987), and β-actin (Santa Cruz Biotechnology Inc.) were used (1:1000), overnight incubation at 4 °C. The blots were then incubated with the corresponding conjugated goat anti-rabbit or goat anti-mouse IgG-horseradish peroxidase (HRP) (1:5000; Santa Cruz Biotechnology Inc.) secondary antibodies, 90 min incubation at room temperature. Immunoreactive proteins were detected using enhanced chemiluminescence (ECL) Western blotting detection system (FUSION SOLO S; Vilber Lourmat, Paris, France). The relative density of the protein bands was measured by ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD).

In this work, the following antibodies were used: primary antibodies: anti-hnRNP UL1 (Abcam #ab68480 or Santa Cruz Biotechnology #sc-393434), anti-FLAG (Sigma #A8592), anti-Actin (MP #691001), anti-Fibrillarin (Santa Cruz Biotechnology #sc-25397), anti-Nucleolin (Abcam #ab22758), anti-FUS (Santa Cruz Biotechnology #sc-47711), anti-γH2A.X (Santa Cruz Biotechnology #sc-517348), anti-RPS6 (Abcam #ab70227), anti-RPS15 (Antikoerper #ABIN2786563), anti-RPA32 (Bethyl Laboratories #A300-245A), anti-pChk1 (Cell Signaling Technology #2341), anti-XRCC1 (Invitrogen #MA5-13412), anti-53BP1 (Abcam #ab175933), anti-Rad50 (Abcam #ab124682), anti-RPA194 (Santa Cruz Biotechnology #sc-46699), normal mouse IgG (Santa Cruz Biotechnology #sc-2025), and anti-digoxygenin-AP Fab fragments (Roche #11093274910); secondary antibodies: goat anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology #sc-516102), goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology #sc-2004), anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific #A21422), anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific #A32723), anti-rabbit Alexa Fluor 555 (Thermo Fisher Scientific #A32732) or anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific #A32731).

Anti-LC3B (#ab48394, Abcam), anti-β-actin (#ab6276, Abcam), anti-HER2 (#2242L Cell Signaling Technology), anti-ATG4B (#A2981, Sigma), anti-ATG4A (#ARP42722_P050, Aviva Systems Biology), anti-ATG4C (#A9482, Sigma), anti-ATG4D (#ABC22, EMD Millipore), anti-Beclin1 (#NB500-249, Novus Biologicals), anti-ATG5 (#2630, Cell Signaling Technology), anti-ATG7 (#NB110-55474, Novus Biologicals), anti-p62 (#P0067, Sigma), goat anti-mouse IgG–horseradish peroxidase (HRP), and goat anti-rabbit IgG–HRP (Santa Cruz Biotechnology) antibodies were used in immunoblotting and immunohistochemistry. The following drugs were used: Bafilomycin A1 (Sigma-Aldrich), and trastuzumab (Herceptin; Genentech Inc., San Francisco, CA).

Anti-LC3B (#ab48394, Abcam), anti-β-actin (#ab6276, Abcam), anti-ATG4B (#A2981, Sigma), anti-p62 (#P0067, Sigma), anti-GABARAP (#ab109364, Abcam), anti-vinculin (#ab129002, Abcam), goat anti-mouse IgG–horseradish peroxidase (HRP), and goat anti-rabbit IgG–HRP (Santa Cruz Biotechnology) antibodies were used in immunoblotting. Wortmannin (Sigma-Aldrich) was used to block autophagosome formation and bafilomycin A1 (Sigma-Aldrich) was used for autophagy flux assays.

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, L-glutamine, antibiotic−antimycotic solution, and trypsin-EDTA were purchased from Invitrogen Co. (Grand Island, NY, USA). Goat antirabbit IgG-HRP, goat anti-mouse IgG-horseradish peroxidase (HRP), mouse anti-goat IgG-HRP, iNOS, IκB-α, p-IκB-α, p50, p65, IL-1β, ERK1/2, p-ERK1/2, and β-actin mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against TNF-α, SHIP1, PTEN, p38, p-p38, SAPK/JNK, p-SAPK/JNK, IRF3, p-IRF3, STAT1, p-STAT1, p-JAK1 rabbit monoclonal antibodies, and JAK1 mouse monoclonal antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). AMV reverse transcriptase, dNTP mixture, random primer, RNasin, and Taq polymerase were purchased from Promega (Madison, WI, USA). Lipopolysaccharide (E. coli O111: B4), polyinosinic-polycytidylic acid (poly (I:C)), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated. Glabralactone (purity > 96% by HPLC analysis) was isolated from an extract of the roots of Angelica sinensis (see Supplementary Material Information).

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), crystal violet, propidium iodide (PI), RNAse, cell lysis buffer, dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors cocktails were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-PARP antibody was acquired from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal anti-β-actin antibody was acquired from Sigma-Aldrich (St. Louis, MO, USA). Anti-phospho-histone H2A.X (Ser139), anti-cyclin B1, goat anti-mouse IgG-horseradish peroxidase (HRP) and goat anti-rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

One-milliliter samples of the cell cultures were taken, pelleted, and resuspended in 1× PBS according to the OD₆₀₀. For samples taken during ΦM1 phage infection, the protein was quantified using a NanoDrop spectrophotometer (ThermoScientific), and equal amounts of protein (150 μg) were resolved by 12% PAGE. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked for 1 h in 1× PBS containing 5% milk powder. Immunodetection of FLAG-tagged ToxN was performed overnight at 4°C in 1× PBS using anti-FLAG M2 antibody (Sigma). Goat anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz) was used as a secondary antibody. Bands were visualized on X-ray film using the SuperSignal West Pico chemiluminescent substrate kit (Pierce). SdhE-FLAG expressed from pMAT7 (54 (link)) was used as a control in the blot tracking ΦM1 infection.