Goat anti mouse igg

For comparison of complement C3 activation in CSF and plasma samples, 20 μl of CSF and 2 μl of plasma samples were loaded on an SDS-PAGE gel. Samples were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then incubated with mouse anti-human C3/C3b/iC3b/C3dg monoclonal antibody (1:1,000, Cedarlane, Ontario, Canada) in TBST (Tris-buffered saline supplemented with Tween 20) containing 5% dry milk at 4 °C overnight. Blots were then washed and incubated with goat anti-mouse IgG (1:15,000, LI-COR Bioscience, Nebraska, USA) diluted in TBST containing 2% dry milk. The membranes were developed with an Odyssey system (Li-COR Bioscience) according to the manufacturer’s protocol.

Verteporfin (VP, Visudyne, Novartis Pharmaceuticals Corp., Basel, Switzerland), a well-characterized PS also serving as an nIR dye with a 700 nm emission peak, and HS201, a novel PS made of VP tethered to an Hsp90 small-molecule inhibitor (HS10), were used for in vitro and in vivo imaging and PDT. The HS201 compound was developed and supplied by the Haystead Lab (Duke University Department of Pharmacology and Cancer Biology) as previously reported [12 (link),13 (link)]. Anti-Hsp90 antibody (clone AC88, Cat# ab13492,) was purchased from Abcam plc. (Cambridge, UK). Secondary antibodies (goat anti-mouse IgG, goat anti-rabbit IgG and donkey anti-mouse IgG antibodies) conjugated with nIR dye, IRDye 800CW (Cat# 926-32210, 926-32211 and 926-32212, respectively), were purchased from LI-COR Inc. (Lincoln, NE, USA). Anti-murine PD-L1 antibody (clone; 10F.9G2, Cat# BE0101) was purchased from Bio X cell (Lebanon, NH, USA) and used for animal treatment.

Ice‐cold radioimmunoprecipitation assay buffer (containing 50 mmol/L Tris‐Hcl, 150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate and 0.1% SDS) was used to extract protein from cardiomyocytes and heart tissue. Then protein was subjected to 10% SDS‐PAGE (50 μg per sample). After transferred onto immobilon membranes (Millipore, Billerica, MA, USA), proteins were incubated with primary antibodies overnight at 4°C. The primary antibodies included the following: ZBTB20 (#ab243143, Abcam, 1:1000 diluted), Bax (#2772), Bcl‐2 (#2870), c‐caspase3 (#9664), T‐caspase3 (#9661), TNFα (#11948), Phospho (P)‐ASK1 (Thr845), total (T)‐ASK1(Thr845)(# #3765), P‐JNK1/2 (#4668p), T‐JNK1/2(#9258), P‐ERK1/2 (#4370P), T‐ERK1/2 (#4695), P‐P38 (#4511P), T‐P38 (#9212P) and GAPDH (#2118, Cell Signaling Technology, 1:1000 diluted), And then incubated with second antibodies of either goat anti‐rabbit IgG (926‐32211; LI‐COR) or goat antimouse IgG (C11026‐03; LI‐COR) for one hour. Analysis and quantification was performed by an Odyssey infrared imaging system (LI‐COR Biosciences). The GAPDH was used as reference.

Proteins were separated by 10% (wt/vol) SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with primary antibodies (all at 1:1,000 dilution) overnight at 4 °C, and subsequently incubated with IRDye 800CW-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (both at 1:10,000 dilution; LI-COR Biosciences, Lincoln, NE, USA) for 1 h. Protein bands were visualized with Odyssey infrared imaging system (LI-COR Biosciences).

Cells were lysed using a buffer with sodium dodecyl sulfate (SDS) (Cell Signaling Technology, Danvers, MA, USA, #9803; following the supplier’s protocol), followed by centrifugation at 12,000× g for 10 min, and the supernatants were recovered as whole-cell extracts. Protein content was measured using the Bradford reagent. Protein extract samples were loaded on 12% polyacrylamide gels (Invitrogen #NP0343BOX, supplied by Fisher Scientific Co., Boston, MA, USA) and electrophoresed for 2 h at 14.5 V/cm. Wet electroblotting transfer was performed for 3 h on ice onto a polyvinylidene difluoride (PVDF) membrane. The primary antibodies included anti-Ape1 (Novus Inc. Guaynabo, PR, USA, #NB100-101; used at 1:1000), anti-DNA2 (Abcam Inc., Waltham, MA, USA, #ab96488; used at 1:500), anti-ExoG (Abcam #ab77736; used at 1:500), anti-Fen1 (Novus NB100-320; used at 1:700), anti-NDUFA9 (specific for a Complex I protein; Abcam ab14713; used at 1:1000), and anti-TFAM (Cell Signaling 7495; used at 1:1000). The secondary antibodies (both used at 1:10,000) were goat anti-rabbit IgG (Licor Inc., Omaha, NB, USA, #925-68021) and goat anti-mouse IgG (Licor 926-32210). The developed blots were placed in distilled water, scanned on a Li-COR Odyssey (Licor Inc. Omaha, NB, USA), and quantified using Image Studio software (version 5.5.4), with n ≥ 3.