Goat anti mouse igg h l hrp conjugate

The protocol was adapted from (Choudhary and Schneiter, 2012 (link)). Briefly, proteins were extracted from three OD₆₀₀ units of yeast cells by NaOH followed by precipitation with 10% TCA. To analyze proteins in the culture supernatant, proteins from 20 ml media of an overnight grown culture were precipitated by 10% TCA.
The primary antibodies used were: anti-HA (rat, 1:2000, Roche #11867423001), c-Myc monoclonal antibody (mouse, 1:5000, Invitrogen #13-2500), monoclonal anti-GFP (mouse, 1:2000, Roche #11814460001), anti-Kar2 (rabbit, 1:5000, Randy Schekman, University of California at Berkeley, CA, USA). As secondary antibodies goat anti-rat IgG antibody, horseradish peroxidase (HRP) conjugate (1:10,000, Merck #AP136P), goat anti-rabbit IgG (H+L)-HRP conjugate (1:10,000, Bio-Rad #1706515) and goat anti-mouse IgG (H+L)-HRP conjugates (1:10,000, Bio-Rad #1706516) were employed.

Gefitinib (AstraZeneca), Phenylarsine Oxide (PAO) (Sigma‐Aldrich), Filipin III (Sigma‐Aldrich), anti‐EGFR (sc‐373746), anti‐EGFR (4267, Cell signaling), anti‐p‐EGFR (2234, Cell signaling), anti‐STAT3 (sc‐8019), anti‐p‐STAT3 (sc‐8059), anti‐ERK (9102, Cell signaling), anti‐p‐ERK (9101, Cell signaling), anti‐EEA1 (610457, BD Biosciences), anti‐PARP (9542, Cell signaling), anti‐c‐Myc (9402, Cell signaling), anti‐β‐actin (A5316), Goat anti‐mouse IgG (H + L)‐HRP conjugate (1706516, Bio‐Rad), Goat anti‐rabbit IgG polyclonal HRP conjugated (ADI‐SAB‐300, Enzo), Alexa Fluor 488 goat anti‐rabbit antibody (A32731, Thermo Fisher Scientific), and Alexa Fluor 594 goat anti‐mouse antibody (A32742, Thermo Fisher Scientific).

Total cellular proteins were extracted from cell lines and PTC tumors using RIPA Lysis Buffer (Thermo Fisher Scientific) with 1% Phenylmethanesulfonyl fluoride (Sigma-Aldrich, Darmstadt, Germany) with or without Protease Inhibitor Cocktail (Sigma-Aldrich, Darmstadt, Germany). For the analysis of phosphorylated AKT, cells were washed once with sodium orthovanadate-containing buffers to inhibit phosphatases prior to RIPA buffer lysis. Thirty micrograms of proteins were separated in Mini-PROTEAN TGX Gels (Bio-Rad Laboratories, Hercules, CA) and transferred to PVDF membranes using Trans-Blot Turbo Transfer Pack (Bio-Rad). Membranes were blocked with 5% non-fat milk diluted in TBST, and then incubated with primary antibodies and secondary antibodies before being imaged with Clarity Max Western ECL Substrate (Bio-Rad, 1705062) and ChemiDoc MP Imaging System (Bio-Rad). The following primary antibodies were used: anti-PLEKHS1 (Novus Biologicals, H00079949-M07) at dilution 1:500, anti-AKT (Cell Signaling Technology, Boston, MA, USA, 9272) and phosphorylated-AKT (Cell Signaling Technology, 4051) at dilution 1:1000, and anti-β-Actin (Santa Cruz, sc-47778, Dallas, TX, USA) at dilution 1:50,000. Secondary antibodies include Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Bio-Rad, 170-6516) and Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (Bio-Rad, 170–6515).

An in-house ELISA assay was designed and accomplished for the verification of anti-CD25 CAR protein reactivity with human CD25 antigen. For this purpose, the CD25 antigen (Novus Biologicals, USA) with the final concentration of 8 μg/mL in 50 mM carbonate/bicarbonate buffer (pH=9.5) was added to each well of 96-well plate and kept at 4°C overnight. After blocking, 100 μL of the isolated anti-CD25 CAR protein with the concentration of 10 μg/mL was added into the test wells as well as the negative (no antigen) control wells. Meanwhile, 100 μL of the daclizumab (Novus Biologicals, USA) with the same concentration was added to the positive control wells and incubated at 37°C for 1 h. Then, the wells were washed three times with PBS. Subsequently, 100 μL of c-Myc mAb-HRP, clone: 9E10 (Invitrogen, USA) in the dilution of 1:750 was added to the test and the negative wells. In addition, 100 μL of Goat anti-mouse IgG (H+L)-HRP conjugate (Bio-Rad, USA) in the dilution of 1:1,000 was added to the positive control wells and incubated at 37°C for 1 h. After washing process, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma-Aldrich, USA) was added to the wells and the plate was kept in the dark for 5 min. Finally, the reaction was stopped by using 2M sulfuric acid and the optical density (OD) was determined by a microplate reader (Hyperion, USA) at 450 nm.

Primary antibodies: The following primary antibodies were used (antigen, dilution, vendor): Sel1L, 1:1000, Abcam ab78298; Actin, 1:4000, Sigma A5441; NPC1, 1:500 western, Abcam ab36983 (discontinued); NPC1, 1:200 imaging, Abcam ab134113; Flag, 1:1000, Sigma F1804; GP78, 1:1000, Cell Signaling 9590; Der-1, 1:1000, Cell Signaling 8897; HRD1, 1:1000, Cell Signaling. D302a; HA, 1:1000, Biolegend 901501; LC3, 1:1000 western, Novus NB600; LC3, 1:200 imaging, Gentex GTX17380; Beclin-1, 1:1000, Santa Cruz H-300; P97, 1:1000, Cell Signaling 2648; Ubiquitin, 1:500, Dako (discontinued); GAPDH, 1:5000, Novus NB600; Vinculin, 1:2000, Sigma V9131; FAM134B, 1:500, Abcam ab151755; FAM134B, 1:500, Proteintech 21537; p62, 1:1000, Sigma P0067; GBA, 1:1000, Sigma G4171; Calreticulin, 1:1000, Cell Signaling Technology 2891, GFP, 1:1000, Abcam ab13970. NPC1, 1:500, Abcam ab134113 was used for Figs. 3b and 5c, Supplementary Fig. S1b, c.
Secondary antibodies: The following secondary antibodies were used (antibody, dilution, vendor): Alexa Fluor 488 Goat anti-rabbit IgG (H+L), 1:500, Invitrogen A11008; Alexa Fluor 594 Fab’2 fragment of goat anti-mouse IgG (H+L), 1:500, Invitrogen A11020; Alexa Fluor 594 goat anti-chicken IgY (H+L), 1:1000, Invitrogen A11042; Goat anti-mouse IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6516; Goat anti-rabbit IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6515.

Microtiter plates (Nerbe Plus GmbH, Winsen/Luhe, Germany) were coated with 100 μL/well of PyV VLPs diluted in the coating buffer (0.05 M sodium carbonate, pH 9.5) at a concentration of 5 μg/mL. The plates were incubated overnight at 4 °C. The coated plates were blocked with 250 μL/well of PBS containing 2% BSA for 1 h at room temperature (RT). The plates were then rinsed twice with PBST (PBS with 0.1 % Tween-20). Antiserum samples, hybridoma growth medium, or PAb were diluted in PBST, added to the wells (100 μL/well), and incubated for 1 h at RT. The plates were then incubated for 1 h with Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Bio-Rad, Hercules, CA, USA) diluted 1:5000 in PBST. The enzymatic reaction was visualised by the addition of 100 μL of “NeA-Blue” TMB solution (Clinical Science Products, Mansfield, MA, USA) to each well. The reaction was stopped by adding 50 μL/well of 1 M sulphuric acid solution. The optical density (OD) was measured at 450 nm (reference filter 620 nm) with a microplate reader (Sunrise Tecan, Männedorf, Switzerland).