Hrp conjugated goat anti mouse igg h l

Protein samples were harvested from cells using the SDS sample buffer (100 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 5% β-mercaptoethanol, and 0.1% bromophenol blue). Samples were heated at 99°C for 10 min prior to being subjected to the immunoblot analysis. The protein samples were then transferred to a PVDF membrane (IPVH00010, pore size 0.45 μm, Merck Millipore). The membrane was blocked using 5% nonfat milk at 25°C for 1 h, followed by the incubation of primary antibodies at 4°C for 16 h. Primary antibodies used were anti-TCF4 (ab217668, 1:1,000, abcam), anti-INTU (ab229243, 1:1,000, abcam), anti-IFT88 (13967-1-AP, 1:1,000, Proteintech) and anti-β-TUBULIN (ab6046, 1:2,000, abcam). The membrane was washed three times with 1× TBST each for 10 min, before being subjected to the incubation of secondary antibodies at 25°C for 1 h. Secondary antibodies used were HRP-conjugated goat anti-rabbit IgG (H + L) (11-035-045, 1:5,000) and HRP-conjugated goat anti-mouse IgG (H + L) (115-035-062, 1:10,000) from Jackson ImmunoResearch. The membrane was washed three times with 1× TBST each for 10 min, prior to the detection of chemiluminescent signal. The signal was developed using Immobilon Forte Western HRP substrate (WBLUF0100, Merck Millipore), and the images were captured and processed using ChemiDoc™ Touch Imaging System (170-8370, Bio-Rad).

Cell lysates and supernatants mixed with the sample buffer were incubated at 95 °C for 10 and 5 min, respectively. After 12% sodium dodecyl sulfate (SDS)-PAGE, separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The PVDF membranes were soaked with 3% skim milk (Becton Dickinson, Franklin Lakes, NJ, USA) in PBS and washed with PBST. Each membrane was incubated with a mouse anti-M1 MAb (APH 6-23-1-6) [34 (link)] and a mouse anti-beta actin antibody (ab6276, Abcam, Cambridge, UK) as primary antibodies and subsequently with HRP-conjugated goat anti-mouse IgG (H + L) (115-035-062, Jackson Immuno Research, West Grove, PA, USA) as a secondary antibody. These antibodies were diluted with PBST containing 1.5% skim milk. The bound antibodies were visualized with Immobilon Western (Merck Millipore, Darmstadt, Germany). The amount of the viral M1 protein was semi-quantified based on the band intensity using an Amersham Imager 600 (GE Healthcare, Little Chalfont, UK).

An ELISpot assay was used to quantify the number of EBOV-GP and RABV-G specific antibody-secreting cells in mouse bone marrow and spleen samples. ELISpot plates were coated with 10 μg/mL EBOV-GP or RABV-G in PBS overnight at 4 °C. Plates were washed with PBS and blocked with goat serum for 1 h at 37 °C followed by 4 °C during the preparation of cells. Bone marrow was harvested from the mouse femurs and tibias. These and the spleens were homogenized followed by red blood cell lysis in ACK buffer; 1.5 × 10⁶ cells were added to antigen-coated plates, serially diluted, and incubated at 37 °C for 16 h. Plates were washed with PBST and incubated in HRP-conjugated goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, Cat# 115-035-003), IgG2c (Cat# 115-035-208), or IgG1 (Cat# 115-035-205) diluted 1:1600 in PBST for 1–2 h at 37 °C. Plates were washed with PBST, followed by PBS, and the TrueBlue peroxidase substrate was added. The reaction was quenched with water, and plates were counted on an AID ELISpot reader.

We used the following primary antibodies: monoclonal antibodies (mAbs) against LC3 (M186-3) and ATG7 (PM039) from MBL, phospho-p70S6 kinase (#9206), p70S6 kinase (Thr389, #9202), phospho-4E-BP1 (Thr37/46, #9459), Myc-Tag (71D10) rabbit mAb (#2278 s) from Cell Signaling Technology. Rac1-102 mAb (#610650) from BD Transduction Laboratories was used to specifically detect the non-glucosylated Rac1 (Non-glu. Rac1), and Rac1 mAb 23A8 (#05-389) from Millipore was used to detect the total Rac1. Monoclonal anti-Flag-HRP (#A8592) was ordered from Sigma. The secondary antibodies HRP-conjugated goat anti-mouse IgG (H+L) (#115-035-003) and HRP-conjugated goat anti-rabbit IgG (H+L) (#111-035-003) were purchased from Jackson Immuno Research. The following chemicals were used: chloroquine (CQ), 3-Methyladenine (3-MA, M9281) and wortmannin (W1628) from Sigma, and 3-⁹⁰ -2,5-diphenyltetrazolium bromide (MTT) from Amresco.

The prepared samples were separated on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gels and transferred to PVDF membranes (Merck, Darmstadt, Hesse, Germany). The membranes were blocked with 0.3% skim milk (FUJIFILM Wako, Osaka, Japan) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and incubated with an anti-c-Myc mouse monoclonal antibody (1:2000, FUJIFILM Wako, 9E10, catalog no. 011-21874) overnight at 4 °C. Then, the membranes were washed three times with TBST and incubated with HRP-conjugated goat anti-mouse IgG (H + L) (1:20,000, Jackson Immunoresearch, West Grove, PA, USA, catalog no. 115-035-003) at RT for 1 h. The membranes were washed three times with TBST and incubated with the ECL solution (Solution 1: 2.5 mM luminol, 4 mM 4-IPBA, and 200 mM Tris-HCl, pH 8.8; Solution 2: 10.6 mM H₂O₂). Equal volumes of each solution were mixed immediately before use. Membranes were imaged using an EZ-capture MG instrument (ATTO, Tokyo, Japan). Quantitative densitometry was used to measure the levels of protein fragments with a CS Analyzer (ATTO).