Hrp conjugated anti mouse secondary antibody

WA-1 or Beta SARS-CoV-2 spike-specific IgG titers were assessed with an antibody titration ELISA as described previously [6 (link)]. Briefly, ELISA plates (Thermo Fisher Scientific, 442404) were coated with 1 µg/mL of SARS-CoV-2 WA-1 S-2P or S-2Pᵦ in PBS (pH 7.4) at 4 °C for 16 h. The plates were washed with PBST three times, then blocked with 5% skim milk in 1 × PBST at RT for 2 h. The sera were diluted by 100-fold in 5% skim milk in PBST. A further serial 4-fold dilution for week-0 and week-2 sera or 6-fold dilution for week-6 sera was applied to the 100-fold dilution preparations. The plates were incubated with diluted sera at RT for 1 h. The HRP-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, 1/2000 dilution) was used to detect the antibody responses. The endpoint titers were calculated as the dilution that yielded an optical density equivalent to 4× background (secondary antibody alone).

All reagents and chemicals were purchased from Sigma-Aldrich unless otherwise specified. Human, adult, normal spleen (NB820-59259) and testis (NB820-59266) lysates and spleen (NBP2-30202), testis (NBP2-75940), skeletal muscle (NBP2-77813) and ovary (NBP2-30190) tissue slides were from Novus Biologicals. Human, adult, normal ovary lysate (HT-406-ZY) was from BioCat. The β-actin (A1978), the vinculin (V9131) and the α-smooth muscle actin (A5228) antibodies were from Sigma-Aldrich, the vWF polyclonal antibody (PA516634), the Alexa Fluor Plus 488 anti-mouse secondary antibody (A32766), the Alexa Fluor 568 anti-rabbit secondary antibody (A10042) and the HRP conjugated anti-mouse secondary antibody (31,432) were from Thermo Fischer Scientific. The mouse IgG1 antibody (554,121) was from BD Biosciences, the calnexin antibody (2679 T) was from Cell Signaling Technology.

ELISA plates (Thermo Fisher, 442404) were coated with 1 µg/ml of SARS-CoV-2 WA1, variant S2P, or HBsAg (ProspecBio, HBS-875) in PBS, pH 7.4 at 4 °C for 16 h^{7 (link)}. Standard washes and blocking steps were done as described above. The plates were blocked for 2 h. The sera were diluted by 100-fold in 5% skim milk in PBST. A serial 4-fold dilution for weeks 0 and 2 sera or 6-fold dilution for week 6 sera was applied to the 100-fold dilution preparations. The plates were incubated with diluted sera at room temperature for 1 h. The HRP-conjugated anti-mouse secondary antibody (Thermo Fisher, G21040, 1/2000) was used to detect the antibody responses. The endpoint titers were calculated as the dilution that yielded an optical density equivalent to 4×background (secondary antibody alone).

A small piece of mycelial fragment from transformant colonies grown on PDHX plates was transferred to 2 mL of MAG medium containing hygromycin (100 µg/mL) in a 24-well microtiter plate and incubated statically in a lighted 30 °C incubator for 3 days till a mycelial mat was observed on the liquid medium. 15 µL of cell-free culture broth (containing secreted proteins) was transferred to microcentrifuge tubes containing 5 µL SDS-PAGE loading buffer and subjected to boiling at 95 °C for 10 min. This protein extract was separated on 4–12% NuPAGE gel in MOPS buffer (200 V for 50 min). Post-separation, proteins were electro-transferred onto PVDF membrane for western blot analysis using an iBlot2 (Thermo Fisher Scientific, Inc. Grand Island, NY, USA). For hybridization of Cel7A protein, a custom generated P. funiculosum anti-Cel7A polyclonal antibody raised in rabbit was used as primary antibody at a dilution of 1:20,000. For detection of eGFP, anti-GFP antibody raised in mouse (Thermo Fisher Scientific Inc., Grand Island, NY, USA) was used as the primary antibody. Detection of cel7A and eGFP proteins was carried out using alkaline phosphatase-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific, Inc. Grand Island, NY, USA), and HRP-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, Inc., Grand Island, NY, USA), respectively.

For characterization of the fibroblasts at the protein level, cells were grown to 90% confluence in 100 mm dishes and lysed in ice‐cold RIPA‐buffer containing protease inhibitors. Cell lysate was collected by centrifugation followed by total protein quantification (BCA kit according to manufacturer's protocol). Expression of eL13 was evaluated in 10 μg of denatured protein samples by Western immunoblotting (WB) using a primary antibody against RPL13 (mouse monoclonal, Santa Cruz Biotechnology, Dallas, TX, USA; #sc‐100829; 1:1,000 dilution) and a HRP‐conjugated anti‐mouse secondary antibody (Thermo Fisher Scientific; #31430, 1:20,000 dilution) according to standard procedures. Acetylated‐tubulin (Sigma‐Aldrich, St. Louis, MO, USA; #T7451, 1:10,000 dilution) was used as a loading control. Further information is available in the Supplemental Materials and Methods.