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Table 5 Denaturation and incubation conditions used in western blot experiments

From: Changes in porcine nutrient transport physiology in response to Ascaris suum infection

Protein

Prep

Denaturation

µg/lane

Primary antibody

Secondary antibody

SGLT1a

AM

40 °C, 15 min

10 µg

1:2000

1:10,000h

pSGLT1b

AM

40 °C, 15 min

10 µg

1:200

1:10,000i

ASCT1c

AM

95 °C, 7 min

10 µg

1:1,000 l

1:2000j

PepT1d

AM

95 °C, 7 min

10 µg

1:500 l

1:2000k

GLUT2e

CM

70 °C, 20 min

10 µg

1:2000

1:20,000h,m

Na+/K+-ATPasef

CM

70 °C, 20 min

10 µg

1:10,000

1:20,000k

Hif-1αg

CY

95 °C, 5 min

10 µg

1:200n

1:2000i

  1. AM apical membrane, CM crude membrane, CY cytosol
  2. aRabbit-anti-SGLT1 (ab14686, Abcam, Cambridge, UK)
  3. bCustom-made (Perbio Science, Bonn, Germany; epitope: KIRKRApSEKELMI)
  4. cRabbit-anti-ASCT1 (#ANT-081, alomone labs, Jerusalem, Israel)
  5. dMouse-anti-PEPT1 (373,742, Santa Cruz, Dallas, TX, USA)
  6. eRabbit-anti-GLUT2 (ABIN310208, Antibodies-online, Aachen, Germany)
  7. fMouse-anti-Na+/K+-ATPase (ALX-804–082, Enzo Life Sciences, Farmingdale, NY, USA)
  8. gRabbit-anti-Hif-1α (#10,006,421, Cayman chemical, Ann Arbor, MI, USA)
  9. hGoat-anti-rabbit HRP (A9169, Sigma-Aldrich, St. Louis, MO, USA)
  10. iGoat-anti-rabbit HRP (#7074, Cell Signaling Technology, Danvers, MA, USA)
  11. jm-IgGκ BP (516,102, Santa Cruz, Dallas, TX, USA)
  12. kGoat-anti-mouse (A2304, Sigma-Aldrich, St. Louis, MO, USA)
  13. lDiluted in BSA (5%) instead of 5% milk/TBST
  14. mDiluted in 2.5% milk/TBST
  15. nDiluted in 2.5% milk/PBS